Engineered imine reductases and methods for the reductive amination of ketone and amine compounds

ABSTRACT

The present application provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered imine reductases, host cells capable of expressing the engineered imine reductases, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.

The present application is a continuation application of co-pending U.S.patent application Ser. No. 16/525,834, filed Jul. 30, 2019, which is aDivisional application of U.S. patent application Ser. No. 15/783,657,filed Oct. 13, 2017, now U.S. Pat. No. 10,407,668, which is a Divisionalapplication of U.S. patent application Ser. No. 14/539,690, filed Nov.12, 2014, now U.S. Pat. No. 9,822,346, which claims priority to U.S.Prov. Pat. Appln. Ser. No. 61/903,772, filed Nov. 13, 2013, U.S. Prov.Pat. Appln. Ser. No. 62/022,315, filed Jul. 9, 2014 and U.S. Prov. Pat.Appln. Ser. No. 62/022,323, filed Jul. 9, 2014, each of which isincorporated by reference in its entirety, for all purposes.

TECHNICAL FIELD

The invention relates to engineered polypeptides having imine reductaseactivity useful for the conversion of various ketone and aminesubstrates to secondary and tertiary amine products.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2-136USP2A_ST25.txt”, a creation date of Jul. 8, 2014,and a size of 2,171 kilobytes. The Sequence Listing filed via EFS-Web ispart of the specification and is incorporated in its entirety byreference herein.

BACKGROUND

Chiral secondary and tertiary amines are important building blocks inpharmaceutical industry. There are no efficient biocatalytic routesknown to produce this class of chiral amine compounds. The existingchemical methods use chiral boron reagents or multi step synthesis.

There are a few reports in the literature of the biocatalytic synthesisof secondary amines. Whole cells of the anaerobic bacteriumAcetobacterium woodii imine reductase activity was reported to reducebenzylidine imines and butylidine imines (Chadha, et al.,Tetrahedron:Asym., 19:93-96 [2008]). Another report uses benzaldehyde orbutyraldehyde and butyl amine or aniline in aqueous medium using wholecells of Acetobacterium woodii (Stephens et al., Tetrahedron 60:753-758[2004]). Streptomyces sp. GF3587 and GF3546 were reported to reduce2-methyl-1-pyrroline stereoselectively (Mitsukara et al., Org. Biomol.Chem. 8:4533-4535 [2010]).

One challenge in developing a biocatalytic route for this type ofreaction is the identification of an enzyme class that could beengineered to provide to carry out such reactions efficiently underindustrially applicable conditions. Opine dehydrogenases are a class ofoxidoreductase that act on CH—NH bonds using NADH or NADPH as co-factor.A native reaction of the opine dehydrogenases is the reductive aminationof α-keto acids with amino acids. At least five naturally occurringgenes having some homology have been identified that encode enzymeshaving the characteristic activity of opine dehydrogenase class. Thesefive enzymes include: opine dehydrogenase from Arthrobactor sp. strain1C (CENDH); octopine dehydrogenase from Pecten maximus (great scallop)(OpDH); ornithine synthase from Lactococcus lactis K1 (CEOS); β-alanineopine dehydrogenase from Cellana grata (BADH); and tauropinedehydrogenase from Suberites domuncula (TauDH). The crystal structure ofthe opine dehydrogenase CENDH has been determined (see Britton et al.,“Crystal structure and active site location ofN-(1-D-carboxyethyl)-L-norvaline dehydrogenase,” Nat. Struct. Biol.5(7): 593-601 (1998)). Another enzyme, N-methyl L-amino aciddehydrogenase from Pseudomonas putida (NMDH) is known to have activitysimilar to opine dehydrogenases, reacting with α-keto acids and alkylamines, but appears to have little or no sequence homology to opinedehydrogenases and amino acid dehydrogenases. NMDH has beencharacterized as belonging to a new superfamily of NAD(P) dependentoxidoreductase (See e.g., U.S. Pat. No. 7,452,704 B2; and Esaki et al.,FEBS J., 272, 1117-1123 [2005]).

There is a need in the art for biocatalysts and processes for usingthem, under industrially applicable conditions, for the synthesis ofchiral secondary and tertiary amines.

SUMMARY

The present invention provides novel biocatalysts and associated methodsto use them for the synthesis of chiral secondary and tertiary amines bydirect reductive amination using an unactivated ketone and anunactivated amine as substrates. The biocatalysts of the invention areengineered polypeptide variants derived by directed evolution of theengineered enzymes of SEQ ID NO:6, which in turn had been generated bydirected evolution of an initial wild-type gene from Arthrobacter sp.strain 1C which encodes an opine dehydrogenase having the amino acidsequence of SEQ ID NO:2. These engineered polypeptides are capable ofcatalyzing the conversion of a ketone (including unactivated ketonesubstrates such as cyclohexanone and 2-pentanone) or aldehyde substrate,and a primary or secondary amine substrate (including unactivated aminesubstrates such as butylamine, aniline, methylamine, and dimethylamine)to form a secondary or tertiary amine product compound. The enzymaticactivity of these engineered polypeptides derived from opinedehydrogenases is referred to as “imine reductase activity,” and theengineered enzymes disclosed herein are also referred to, as “iminereductases” or “IREDs.” The general imine reductase activity of theIREDs is illustrated below in Scheme 1.

The engineered polypeptides having imine reductase activity of thepresent invention can accept a wide range of substrates. Accordingly, inthe biocatalytic reaction of Scheme 1, the R¹ and R² groups of thesubstrate of formula (I) are independently selected from a hydrogenatom, or optionally substituted alkyl, alkenyl, alkynyl, alkoxy,carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl,arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; and the R³and R⁴ groups of the substrate of formula (II) are independentlyselected from a hydrogen atom, and optionally substituted alkyl,alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl,alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl,heteroaryl, and heteroarylalkyl, with the proviso that both R³ and R⁴cannot be hydrogen. Optionally, either or both of the R¹ and R² groupsof the substrate of formula (I) and the R³ and R⁴ groups of thesubstrate of formula (II), can be linked to form a 3-membered to10-membered ring. Further, the biocatalytic reaction of Scheme 1 can bean intramolecular reaction wherein at least one of the R¹ and R² groupsof the compound of formula (I) is linked to at least one of the R³ andR⁴ groups of the compound of formula (II). Also, either or both of thecarbon atom and/or the nitrogen indicated by * in the product compoundof formula (III) can be chiral. As described further herein, theengineered polypeptides having imine reductase activity exhibitstereoselectivity, thus, an imine reductase reaction of Scheme 1 can beused to establish one, two, or more, chiral centers of a productcompound of formula (III) in a single biocatalytic reaction.

In some embodiments, the present invention provides an engineeredpolypeptide comprising an amino acid sequence having at least 80%sequence identity to an amino acid reference sequence of SEQ ID NOS:2,4, or 6, and further comprising one or more amino acid residuedifferences as compared to the reference amino sequence, wherein theengineered polypeptide has imine reductase activity. In some embodimentsof the engineered polypeptide, the imine reductase activity is theactivity of Scheme 1, optionally, a reaction as disclosed in Table 2.

Additionally, as noted above, the crystal structure of the opinedehydrogenase CENDH has been determined (See e.g., Britton et al.,“Crystal structure and active site location ofN-(1-D-carboxyethyl)-L-norvaline dehydrogenase,” Nat. Struct. Biol. 5:593-601 [1998]). Accordingly, this correlation of the various amino aciddifferences and functional activity disclosed herein along with theknown three-dimensional structure of the wild-type enzyme CENDH canprovide the ordinary artisan with sufficient information to rationallyengineer further amino acid residue changes to the polypeptides providedherein (and to homologous opine dehydrogenase enzymes including OpDH,BADH, CEOS, and TauDH), and retain or improve on the imine reductaseactivity or stability properties. In some embodiments, it iscontemplated that such improvements can include engineering theengineered polypeptides of the present invention to have imine reductaseactivity with a range of substrates and provide a range of products asdescribed in Scheme 1.

In some embodiments, the present invention provides an engineeredpolypeptide engineered polypeptide comprising an amino acid sequencewith at least 80% sequence identity to a reference sequence of SEQ IDNO:6 and at least one of the following features:

(i) a residue difference as compared to the reference sequence of SEQ IDNO:6 at a position selected from X12, X18, X26, X27, X57, X65, X87, X93,X96, X126, X138, X140, X142, X159, X170, X175, X177, X195, X200, X221,X234, X241, X242, X253, X254, X257, X262, X263, X267, X272, X276, X277,X278, X281, X282, X291, and X352, optionally wherein the residuedifference at the position is selected from X12M, X18G, X26M/V, X27S,X57D/L/V, X65I/V, X87A, X93G/Y, X96C, X126S, X138L, X140M, X142A,X159C/L/Q/V, X170F/K/R/S, X175R, X177R, X195S, X200S, X221F, 234C/L,X241K, X242C/L, X253K/N, X254R, X257Q, X262F/G/P/V,X263C/D/E/H/M/L/M/N/P/Q/V, X267E/G/H/I/N/S, X272D, X276L, X277H/L,X278E/H/K/N/R/S/W, X281A, X282A/R, X291E, and X352Q;

(ii) a residue difference as compared to the reference sequence of SEQID NO:6 selected from X20V, X29K, X37P, X74W, X82C/T, X94N, X108S,X111A/H, X141M/N, X143F/L/Y, X153F, X154C/D/G/K/L/N/S/T/V,X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V, X163V, X197V, X201I,X220C/K/Q, X223S, X256A/E/I/L/S/T, X259C/R, X260A/D/N/Q/V/Y,X261E/F/H/L/P/Q/Y, X264V, X270L, X273C, X274L/S, X279T, X284C/F/H/P/Q/S,X292E/P, and X295F; and/or

(iii) two or more residue differences as compared to the referencesequence of SEQ ID NO:6 selected from X82P, X141W, X153Y, X154F,X259I/L/M, X274L/M, X283V, and X296N/V;

wherein the polypeptide has imine reductase activity.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence comprising at least oneresidue difference as compared to the reference sequence of SEQ ID NO:6selected from X12M, X37P, X82T, X111A, X154S, X156N/M, X223S, X256E,X260D, X261H, X262P, X263C/E/Q, X267G, X277L, X281A, X284P/S, and X292E.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence comprising at least oneresidue difference as compared to the reference sequence of SEQ ID NO:6selected from X93G/Y, X94N, X96C, X111A/H, X142A, X159L, X163V, X256E,X259R, X273C, and X284P/S.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence comprising at least tworesidue differences as compared to the reference sequence of SEQ ID NO:6selected from X82P, X141W, X143W, X153Y, X154F/Q/Y, X256V, X259I/L/M/T,X260G, X261R, X265L, X273W, X274M, X277A/I, X279L, X283V, X284L, X296N,X326V. In some embodiments, the at least two residue differences areselected from X82P, X141W, X153Y, X154F, X259I/L/M, X274L/M, X283V, andX296N/V.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence comprising at least acombination of residue differences as compared to the reference sequenceof SEQ ID NO:6 selected from:

(a) X153Y, and X283V;

(b) X141W, X153Y, and X283V;

(c) X141W, X153Y, X274L/M, and X283V;

(d) X141W, X153Y, X154F, X274L/M, and X283V;

(e) X141W, X153Y, X154F, and X283V;

(f) X141W, X153Y, X283V, and X296N/V;

(g) X141W, X153Y, X274L/M, X283V, and X296N/V:

(h) X111A, X153Y, X256E, X274M, and X283V;

(i) X111A, X141W, X153Y, X273C, X274M, X283V, and X284S;

(j) X111A, X141W, X153Y, X273C, and X283V;

(k) X111A, X141W, X153Y, X154F, X256E, X274M, X283V, X284S, and X296N;

(l) X111A, X141W, X153Y, X256E, X273W, X274L, X283V, X284S, and X296N;

(m) X111H, X141W, X153Y, X273W, X274M, X284S, and X296N;

(n) X111H, X141W, X153Y, X154F, X273W, X274L, X283V, X284S, and X296N;

(o) X82P, X141W, X153Y, X256E, X274M, and X283V;

(p) X82P, X111A, X141W, X153Y, X256E, X274M, X283V, M284S, and E296V;

(q) X94N, X143W, X159L, X163V, X259M, and X279L;

(r) X141W, X153Y, X154F, and X256E; and

(s) X153Y, X256E, and X274M.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence comprising at least one of theabove combinations of amino acid residue differences (a)−(s), andfurther comprises at least one residue difference as compared to thereference sequence of SEQ ID NO:6 selected from X12M, X18G, X20V,X26M/V, X27S, X29K, X37P, X57D/L/V, X65I/V, X74W, X82C/T, X87A, X93G/Y,X94N, X96C, X108S, X111A/H, X126S, X138L, X140M, X141M/N, X142A,X143F/L/Y, X153E/F, X154C/D/G/K/L/N/S/T/V, X156H/L/N/M/R, X157F/Q/T/Y,X158I/L/R/S/T/V, X159C/L/Q/V, X163V, X170F/K/R/S, X175R, X177R, X195S,X197V, X200S, X201I, X220C/K/Q, X221F, X223S, X234V/C/L, X241K, X242C/L,X253K/N, X254R, X256A/E/I/L/S/T, X257Q, X259C/R, X260A/D/N/Q/V/Y,X261E/F/H/L/P/Q/Y, X262P, X262F/G/V, X263C/D/E/H/IM/L/M/N/P/Q/V, X264V,X267E/G/H/I/N/S, X270L, X272D, X273C, X274L/S, X276L, X277H/L,X278E/H/K/N/R/S/W, X279T, X281A, X282A/R, X284C/F/H/P/Q/S, X291E,X292E/P, X295F, and X352Q.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises the amino acid sequence comprises the combination ofresidue differences X111A, X141W, X153Y, X154F, X256E, X274M, X283V,X284S, and X296N and at least a residue difference or a combination ofresidue differences as compared to the reference sequence of SEQ ID NO:6selected from:

(a) X156N;

(b) X37P, X82T, and X156N;

(c) X37P, X82T, X156N, and X259I;

(d) X259L/M;

(e) X82T, X156N, X223S, X259L, X267G, and X281A;

(f) X263C;

(g) X12M, X261H, X263C, X277L, and X292E;

(h) X154S; and

(i) X154S, X156M, X260D, X261H, X262P, X263E, and X284P.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence having 70%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or greater identity to a sequence of even-numberedsequence identifiers SEQ ID NOS:8-924. In some embodiments, thereference sequence is selected from SEQ ID NOS:6, 12, 84, 92, 146, 162,198, 228, 250, 324, 354, 440, 604, 928, 944, 1040, and 1088.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence having 70%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or greater identity to a sequence of even-numberedsequence identifiers SEQ ID NOS:6-924, wherein the amino acid sequencecomprises an amino acid residue difference as disclosed above (andelsewhere herein) but which does not include a residue difference ascompared to the reference sequence of SEQ ID NO:6 at one or more residuepositions selected from X29, X137, X157, X184, X197, X198, X201, X220,X232, X261, X266, X279, X280, X287, X288, X293, X295, X311, X324, X328,X332, and X353.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence having 70%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or greater identity to a sequence of even-numberedsequence identifiers SEQ ID NOS:6-924, wherein the amino acid sequencecomprises an amino acid residue difference as disclosed above (andelsewhere herein), wherein the amino acid sequence further comprises aresidue difference as compared to the reference sequence of SEQ ID NO:6selected from: X4H/L/R, X5T, X14P, X20T, X29R/T, X37H, X67A/D, X71C/V,X74R, X82P, X94K/R/T, X97P, X100W, X111M/Q/R/S, X124L/N, X136G, X137N,X141W, X143W, X149L, X153E/V/Y, X154F/M/Q/Y, X156G/I/Q/S/T/V,X157D/H/L/M/N/R, X158K, X160N, X163T, X177C/H, X178E, X183C, X184K/Q/R,X185V, X186K/R, X197I/P, X198A/E/H/P/S, X201L, X220D/H, X223T, X226L,X232G/A/R, X243G, X246W, X256V, X258D, X259E/H/I/L/M/S/T/V/W, X260G,X261A/G/M/R/S/T, X265G/L/Y, X266T, X270G, X273W, X274M, X277A/I,X279F/L/V/Y, X280L, X283M/V, X284K/L/M/Y, X287S/T, X288G/S,X292C/G/I/P/S/TN/Y, X293H/I/K/L/N/Q/TN, X294A/I/V, X295R/S, X296L/N/V/W,X297A, X308F, X311C/T/V, X323C/I/M/TN, X324L/T, X326V, X328A/G/E, X332V,X353E, and X356R.

In another aspect, the present invention provides polynucleotidesencoding any of the engineered polypeptides having imine reductaseactivity disclosed herein. Exemplary polynucleotide sequences areprovided in the Sequence Listing incorporated by reference herein andinclude the sequences of odd-numbered sequence identifiers SEQ ID NOS:7-923.

In another aspect, the polynucleotides encoding the engineeredpolypeptides having imine reductase activity of the invention can beincorporated into expression vectors and host cells for expression ofthe polynucleotides and the corresponding encoded polypeptides. As such,in some embodiments, the present invention provides methods of preparingthe engineered polypeptides having imine reductase activity by culturinga host cell comprising the polynucleotide or expression vector capableof expressing an engineered polypeptide of the invention underconditions suitable for expression of the polypeptide. In someembodiments, the method of preparing the imine reductase polypeptide cancomprise the additional step of isolating the expressed polypeptide.

In some embodiments, the present invention also provides methods formanufacturing further engineered polypeptides having imine reductaseactivity, wherein the method can comprise: (a) synthesizing apolynucleotide encoding a reference amino acid sequence selected fromthe even-numbered sequence identifiers of SEQ ID NOS:8-924, and furtheraltering this reference sequence to include one or more amino acidresidue differences as compared to the selected reference sequence atresidue positions disclosed above and elsewhere herein. For example, thespecific positions and amino acid residue differences can be selectedfrom X12M, X18G, X20V, X26M/V, X27S, X29K, X37P, X57D/L/V, X65I/V, X74W,X82C/T, X87A, X93G/Y, X94N, X96C, X108S, X111A/H, X126S, X138L, X140M,X141M/N, X142A, X143F/L/Y, X153E/F, X154C/D/G/K/L/N/S/T/V,X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V, X159C/L/Q/V, X163V,X170F/K/R/S, X175R, X177R, X195S, X197V, X200S, X201I, X220C/K/Q, X221F,X223S, X234V/C/L, X241K, X242C/L, X253K/N, X254R, X256A/E/I/L/S/T,X257Q, X259C/R, X260A/D/N/Q/V/Y, X261E/F/H/L/P/Q/Y, X262P, X262F/G/V,X263C/D/E/H/M/L/M/N/P/Q/V, X264V, X267E/G/H/I/N/S, X270L, X272D, X273C,X274L/S, X276L, X277H/L, X278E/H/K/N/R/S/W, X279T, X281A, X282A/R,X284C/F/H/P/Q/S, X291E, X292E/P, X295F, and X352Q. As further providedin the detailed description, additional variations can be incorporatedduring the synthesis of the polynucleotide to prepare engineered iminereductase polypeptides with corresponding differences in the expressedamino acid sequences.

In some embodiments, the engineered polypeptides having imine reductaseactivity of the present invention can be used in a biocatalytic processfor preparing a secondary or tertiary amine product compound of formula(III),

wherein, R¹ and R² groups are independently selected from optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl; and optionally R¹ andR² are linked to form a 3-membered to 10-membered ring; R³ and R⁴ groupsare independently selected from a hydrogen atom, and optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl, with the proviso thatboth R³ and R⁴ cannot be hydrogen; and optionally R³ and R⁴ are linkedto form a 3-membered to 10-membered ring; and optionally, the carbonatom and/or the nitrogen indicated by * is chiral. The process comprisescontacting a compound of formula (I),

wherein R¹, and R² are as defined above; and a compound of formula (II),

wherein R³, and R⁴ are as defined above; with an engineered polypeptidehaving imine reductase activity in presence of a cofactor under suitablereaction conditions.

In some embodiments of the above biocatalytic process, the engineeredpolypeptide having imine reductase activity is derived via directedevolution of the engineered reference polypeptide of SEQ ID NO:6 (whichwas derived from the opine dehydrogenase from Arthrobacter sp. strain 1Cof SEQ ID NO:2). Any of the engineered imine reductases described herein(and exemplified by the engineered imine reductase polypeptides of evennumbered sequence identifiers SEQ ID NOS:8-924) can be used in thebiocatalytic processes for preparing a secondary or tertiary aminecompound of formula (III).

In some embodiments of the process for preparing a product compound offormula (III) using an engineered imine reductase of the presentinvention, the process further comprises a cofactor regeneration systemcapable of converting NADP⁺ to NADPH, or NAD to NADH. In someembodiments, the cofactor recycling system comprises formate and formatedehydrogenase (FDH), glucose and glucose dehydrogenase (GDH),glucose-6-phosphate and glucose-6-phosphate dehydrogenase, a secondaryalcohol and alcohol dehydrogenase, or phosphite and phosphitedehydrogenase. In some embodiments, the process can be carried out,wherein the engineered imine reductase is immobilized on a solidsupport.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly indicates otherwise. Thus, for example, reference to “apolypeptide” includes more than one polypeptide. Similarly, “comprise,”“comprises,” “comprising” “include,” “includes,” and “including” areinterchangeable and not intended to be limiting. It is to be understoodthat where descriptions of various embodiments use the term“comprising,” those skilled in the art would understand that in somespecific instances, an embodiment can be alternatively described usinglanguage “consisting essentially of” or “consisting of.” It is to befurther understood that where descriptions of various embodiments usethe term “optional” or “optionally” the subsequently described event orcircumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not. It is to be understood that both the foregoing generaldescription, and the following detailed description are exemplary andexplanatory only and are not restrictive of this invention. The sectionheadings used herein are for organizational purposes only and not to beconstrued as limiting the subject matter described.

Abbreviations

The abbreviations used for the genetically encoded amino acids areconventional and are as follows:

Three- One-Letter Amino Acid Letter Abbreviation Alanine Ala A ArginineArg R Asparagine Asn N Aspartate Asp D Cysteine Cys C Glutamate Glu EGlutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleotides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

Definitions

In reference to the present invention, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings:

“Protein”, “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Polynucleotide” or “nucleic acid′ refers to two or more nucleosidesthat are covalently linked together. The polynucleotide may be whollycomprised ribonucleosides (i.e., an RNA), wholly comprised of 2′deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2′deoxyribonucleosides. While the nucleosides will typically be linkedtogether via standard phosphodiester linkages, the polynucleotides mayinclude one or more non-standard linkages. The polynucleotide may besingle-stranded or double-stranded, or may include both single-strandedregions and double-stranded regions. Moreover, while a polynucleotidewill typically be composed of the naturally occurring encodingnucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), itmay include one or more modified and/or synthetic nucleobases, such as,for example, inosine, xanthine, hypoxanthine, etc. Preferably, suchmodified or synthetic nucleobases will be encoding nucleobases.

“Opine dehydrogenase activity,” as used herein, refers to an enzymaticactivity in which a carbonyl group of a 2-ketoacid (e.g., pyruvate) andan amino group of a neutral L-amino acid (e.g., L-norvaline) areconverted to a secondary amine dicarboxylate compound (e.g., such asN-[1-(R)-(carboxy)ethyl]-(S)-norvaline).

“Opine dehydrogenase,” as used herein refers to an enzyme having opinedehydrogenase activity. Opine dehydrogenase includes but is not limitedto the following naturally occurring enzymes: opine dehydrogenase fromArthrobacter sp. strain 1C (CENDH) (SEQ ID NO:2); octopine dehydrogenasefrom Pecten maximus (OpDH) (SEQ ID NO:102); ornithine synthase fromLactococcus lactis K1 (CEOS) (SEQ ID NO:104); N-methyl L-amino aciddehydrogenase from Pseudomonas putida (NMDH) (SEQ ID NO:106);β-alanopine dehydrogenase from Cellana grata (BADH) (SEQ ID NO:108);tauropine dehydrogenase from Suberites domuncula (TauDH) (SEQ IDNO:110); saccharopine dehydrogenase from Yarrowia lipolytica (SacDH)(UniProtKB entry: P38997, entry name: LYS1_YARLI); and D-nopalinedehydrogenase from Agrobacterium tumefaciens (strain T37) (UniProtKBentry: P00386, entry name: DHNO_AGRT7).

“Imine reductase activity,” as used herein, refers to an enzymaticactivity in which a carbonyl group of a ketone or aldehyde and an aminogroup a primary or secondary amine (wherein the carbonyl and aminogroups can be on separate compounds or the same compound) are convertedto a secondary or tertiary amine product compound, in the presence ofco-factor NAD(P)H, as illustrated in Scheme 1.

“Imine reductase” or “IRED,” as used herein, refers to an enzyme havingimine reductase activity. It is to be understood that imine reductasesare not limited to engineered polypeptides derived from the wild-typeopine dehydrogenase from Arthrobacter sp. strain 1C, but may includeother enzymes having imine reductase activity, including engineeredpolypeptides derived from other opine dehydrogenase enzymes, such asoctopine dehydrogenase from Pecten maximus (OpDH), ornithine synthasefrom Lactococcus lactis K1 (CEOS), β-alanopine dehydrogenase fromCellana grata (BADH), tauropine dehydrogenase from Suberites domuncula(TauDH); and N-methyl L-amino acid dehydrogenase from Pseudomonas putida(NMDH); or an engineered enzyme derived from a wild-type enzyme havingimine reductase activity. Imine reductases as used herein includenaturally occurring (wild-type) imine reductase as well as non-naturallyoccurring engineered polypeptides generated by human manipulation.

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” or “engineered” or “non-naturally occurring” when usedwith reference to, e.g., a cell, nucleic acid, or polypeptide, refers toa material, or a material corresponding to the natural or native form ofthe material, that has been modified in a manner that would nototherwise exist in nature, or is identical thereto but produced orderived from synthetic materials and/or by manipulation usingrecombinant techniques. Non-limiting examples include, among others,recombinant cells expressing genes that are not found within the native(non-recombinant) form of the cell or express native genes that areotherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence for optimal alignment of the two sequences. Thepercentage may be calculated by determining the number of positions atwhich the identical nucleic acid base or amino acid residue occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Alternatively, the percentage may becalculated by determining the number of positions at which either theidentical nucleic acid base or amino acid residue occurs in bothsequences or a nucleic acid base or amino acid residue is aligned with agap to yield the number of matched positions, dividing the number ofmatched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Those of skill in the art appreciate that there aremany established algorithms available to align two sequences. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch,1970, J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package), or by visualinspection (see generally, Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1995Supplement) (Ausubel)). Examples of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, NucleicAcids Res. 3389-3402, respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplarydetermination of sequence alignment and % sequence identity can employthe BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. In some embodiments, a “referencesequence” can be based on a primary amino acid sequence, where thereference sequence is a sequence that can have one or more changes inthe primary sequence. For instance, a “reference sequence based on SEQID NO:4 having at the residue corresponding to X14 a valine” or X14Vrefers to a reference sequence in which the corresponding residue at X14in SEQ ID NO:4, which is a tyrosine, has been changed to valine.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent identity and 89 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 residue positions, frequentlyover a window of at least 30-50 residues, wherein the percentage ofsequence identity is calculated by comparing the reference sequence to asequence that includes deletions or additions which total 20 percent orless of the reference sequence over the window of comparison. Inspecific embodiments applied to polypeptides, the term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80 percent sequence identity, preferably at least 89 percentsequence identity, at least 95 percent sequence identity or more (e.g.,99 percent sequence identity). Preferably, residue positions which arenot identical differ by conservative amino acid substitutions.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredimine reductase, can be aligned to a reference sequence by introducinggaps to optimize residue matches between the two sequences. In thesecases, although the gaps are present, the numbering of the residue inthe given amino acid or polynucleotide sequence is made with respect tothe reference sequence to which it has been aligned.

“Amino acid difference” or “residue difference” refers to a change inthe amino acid residue at a position of a polypeptide sequence relativeto the amino acid residue at a corresponding position in a referencesequence. The positions of amino acid differences generally are referredto herein as “Xn,” where n refers to the corresponding position in thereference sequence upon which the residue difference is based. Forexample, a “residue difference at position X25 as compared to SEQ IDNO:2” refers to a change of the amino acid residue at the polypeptideposition corresponding to position 25 of SEQ ID NO:2. Thus, if thereference polypeptide of SEQ ID NO:2 has a valine at position 25, then a“residue difference at position X25 as compared to SEQ ID NO:2” an aminoacid substitution of any residue other than valine at the position ofthe polypeptide corresponding to position 25 of SEQ ID NO:2. In mostinstances herein, the specific amino acid residue difference at aposition is indicated as “XnY” where “Xn” specified the correspondingposition as described above, and “Y” is the single letter identifier ofthe amino acid found in the engineered polypeptide (i.e., the differentresidue than in the reference polypeptide). In some embodiments, theremore than one amino acid can appear in a specified residue position, thealternative amino acids can be listed in the form XnY/Z, where Y and Zrepresent alternate amino acid residues. In some instances (e.g., inTables 3A, 3B, 3C, 3D and 3E), the present invention also providesspecific amino acid differences denoted by the conventional notation“MB”, where A is the single letter identifier of the residue in thereference sequence, “n” is the number of the residue position in thereference sequence, and B is the single letter identifier of the residuesubstitution in the sequence of the engineered polypeptide. Furthermore,in some instances, a polypeptide of the present invention can includeone or more amino acid residue differences relative to a referencesequence, which is indicated by a list of the specified positions wherechanges are made relative to the reference sequence. The presentinvention includes engineered polypeptide sequences comprising one ormore amino acid differences that include either/or both conservative andnon-conservative amino acid substitutions.

“Conservative amino acid substitution” refers to a substitution of aresidue with a different residue having a similar side chain, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. By way of example and not limitation, an amino acid with analiphatic side chain may be substituted with another aliphatic aminoacid, e.g., alanine, valine, leucine, and isoleucine; an amino acid withhydroxyl side chain is substituted with another amino acid with ahydroxyl side chain, e.g., serine and threonine; an amino acid havingaromatic side chains is substituted with another amino acid having anaromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, andhistidine; an amino acid with a basic side chain is substituted withanother amino acid with a basic side chain, e.g., lysine and arginine;an amino acid with an acidic side chain is substituted with anotheramino acid with an acidic side chain, e.g., aspartic acid or glutamicacid; and a hydrophobic or hydrophilic amino acid is replaced withanother hydrophobic or hydrophilic amino acid, respectively. Exemplaryconservative substitutions are provided in Table 1 below.

TABLE 1 Residue Possible Conservative Substitutions A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) N, Q, S, T Other polar H, Y, W, F Other aromatic (H, Y, W, F) C,P None

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine), (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered imine reductase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. In some embodiments,the improved engineered imine reductase enzymes comprise insertions ofone or more amino acids to the naturally occurring polypeptide havingimine reductase activity as well as insertions of one or more aminoacids to other improved imine reductase polypeptides. Insertions can bein the internal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion proteins as is knownin the art. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the naturally occurringpolypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length imine reductasepolypeptide, for example the polypeptide of SEQ ID NO:2 or engineeredimine reductase of SEQ ID NO:96.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The engineered imine reductase enzymes may be present withina cell, present in the cellular medium, or prepared in various forms,such as lysates or isolated preparations. As such, in some embodiments,the engineered imine reductase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure imine reductase compositionwill comprise about 60% or more, about 70% or more, about 80% or more,about 90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedengineered imine reductase polypeptide is a substantially purepolypeptide composition.

“Stereoselective” refers to a preference for formation of onestereoisomer over another in a chemical or enzymatic reaction.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to a chemical or enzymatic reaction thatis capable of converting a substrate or substrates, e.g., substratecompounds (1e) and (2b), to the corresponding amine product, e.g.,compound (3i), with at least about 85% stereomeric excess.

“Improved enzyme property” refers to an imine reductase polypeptide thatexhibits an improvement in any enzyme property as compared to areference imine reductase. For the engineered imine reductasepolypeptides described herein, the comparison is generally made to thewild-type enzyme from which the imine reductase is derived, although insome embodiments, the reference enzyme can be another improvedengineered imine reductase. Enzyme properties for which improvement isdesirable include, but are not limited to, enzymatic activity (which canbe expressed in terms of percent conversion of the substrate), thermostability, solvent stability, pH activity profile, cofactorrequirements, refractoriness to inhibitors (e.g., substrate or productinhibition), stereospecificity, and stereoselectivity (includingenantioselectivity).

“Increased enzymatic activity” refers to an improved property of theengineered imine reductase polypeptides, which can be represented by anincrease in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount of iminereductase) as compared to the reference imine reductase enzyme.Exemplary methods to determine enzyme activity are provided in theExamples. Any property relating to enzyme activity may be affected,including the classical enzyme properties of K_(m), V_(max) or k_(cat),changes of which can lead to increased enzymatic activity. Improvementsin enzyme activity can be from about 1.2 times the enzymatic activity ofthe corresponding wild-type enzyme, to as much as 2 times, 5 times, 10times, 20 times, 25 times, 50 times or more enzymatic activity than thenaturally occurring or another engineered imine reductase from which theimine reductase polypeptides were derived Imine reductase activity canbe measured by any one of standard assays, such as by monitoring changesin properties of substrates, cofactors, or products. In someembodiments, the amount of products generated can be measured by LiquidChromatography-Mass Spectrometry (LC-MS). Comparisons of enzymeactivities are made using a defined preparation of enzyme, a definedassay under a set condition, and one or more defined substrates, asfurther described in detail herein. Generally, when lysates arecompared, the numbers of cells and the amount of protein assayed aredetermined as well as use of identical expression systems and identicalhost cells to minimize variations in amount of enzyme produced by thehost cells and present in the lysates.

“Conversion” refers to the enzymatic conversion of the substrate(s) tothe corresponding product(s). “Percent conversion” refers to the percentof the substrate that is converted to the product within a period oftime under specified conditions. Thus, the “enzymatic activity” or“activity” of a imine reductase polypeptide can be expressed as “percentconversion” of the substrate to the product.

“Thermostable” refers to a imine reductase polypeptide that maintainssimilar activity (more than 60% to 80% for example) after exposure toelevated temperatures (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the wild-type enzyme.

“Solvent stable” refers to an imine reductase polypeptide that maintainssimilar activity (more than e.g., 60% to 80%) after exposure to varyingconcentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol,dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran,acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for aperiod of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme.

“Thermo- and solvent stable” refers to an imine reductase polypeptidethat is both thermostable and solvent stable.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci USA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad.Sci USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychliket al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, NucleicAcids Res 19:698); Sambrook et al., supra); Suggs et al., 1981, InDevelopmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered imine reductase enzyme of the present invention.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding theimine reductase enzymes may be codon optimized for optimal productionfrom the host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present invention. Each control sequence maybe native or foreign to the nucleic acid sequence encoding thepolypeptide. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

“Suitable reaction conditions” refer to those conditions in thebiocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, cofactor loading, temperature, pH, buffers,co-solvents, etc.) under which an imine reductase polypeptide of thepresent invention is capable of converting a substrate compound to aproduct compound (e.g., conversion of compound (2) to compound (1)).Exemplary “suitable reaction conditions” are provided in the presentinvention and illustrated by the Examples.

“Cofactor regeneration system” or “cofactor recycling system” refers toa set of reactants that participate in a reaction that reduces theoxidized form of the cofactor (e.g., NADP⁺ to NADPH). Cofactors oxidizedby the imine reductase catalyzed reductive amination of the ketonesubstrate are regenerated in reduced form by the cofactor regenerationsystem. Cofactor regeneration systems comprise a stoichiometricreductant that is a source of reducing hydrogen equivalents and iscapable of reducing the oxidized form of the cofactor. The cofactorregeneration system may further comprise a catalyst, for example anenzyme catalyst that catalyzes the reduction of the oxidized form of thecofactor by the reductant. Cofactor regeneration systems to regenerateNADH or NADPH from NAD⁺ or NADP⁺, respectively, are known in the art andmay be used in the methods described herein.

“Formate dehydrogenase” and “FDH” are used interchangeably herein torefer to an NAD⁺ or NADP⁺-dependent enzyme that catalyzes the conversionof formate and NAD⁺ or NADP⁺ to carbon dioxide and NADH or NADPH,respectively.

“Loading”, such as in “compound loading” or “enzyme loading” or“cofactor loading” refers to the concentration or amount of a componentin a reaction mixture at the start of the reaction.

“Substrate” in the context of a biocatalyst mediated process refers tothe compound or molecule acted on by the biocatalyst. For example, animine reductase biocatalyst used in the reductive amination processesdisclosed herein there is a ketone (or aldehyde) substrate of formula(I), such as cyclohexanone, and an amine substrate of formula (II), suchas butylamine.

“Product” in the context of a biocatalyst mediated process refers to thecompound or molecule resulting from the action of the biocatalyst. Forexample, an exemplary product for an imine reductase biocatalyst used ina process disclosed herein is a secondary or tertiary amine compound,such as a compound of formula (III).

“Alkyl” refers to saturated hydrocarbon groups of from 1 to 18 carbonatoms inclusively, either straight chained or branched, more preferablyfrom 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbonatoms inclusively. An alkyl with a specified number of carbon atoms isdenoted in parenthesis, e.g., (C₁-C₆)alkyl refers to an alkyl of 1 to 6carbon atoms.

“Alkylene” refers to a straight or branched chain divalent hydrocarbonradical having from 1 to 18 carbon atoms inclusively, more preferablyfrom 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbonatoms inclusively.

“Alkenyl” refers to groups of from 2 to 12 carbon atoms inclusively,either straight or branched containing at least one double bond butoptionally containing more than one double bond.

“Alkenylene” refers to a straight or branched chain divalent hydrocarbonradical having 2 to 12 carbon atoms inclusively and one or morecarbon-carbon double bonds, more preferably from 2 to 8 carbon atomsinclusively, and most preferably 2 to 6 carbon atoms inclusively.

“Alkynyl” refers to groups of from 2 to 12 carbon atoms inclusively,either straight or branched containing at least one triple bond butoptionally containing more than one triple bond, and additionallyoptionally containing one or more double bonded moieties.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 12 carbon atomsinclusively having a single cyclic ring or multiple condensed ringswhich can be optionally substituted with from 1 to 3 alkyl groups.Exemplary cycloalkyl groups include, but are not limited to, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl,1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and thelike, or multiple ring structures, including bridged ring systems, suchas adamantyl, and the like.

“Cycloalkylalkyl” refers to an alkyl substituted with a cycloalkyl,i.e., cycloalkyl-alkyl-groups, preferably having from 1 to 6 carbonatoms inclusively in the alkyl moiety and from 3 to 12 carbon atomsinclusively in the cycloalkyl moiety. Such cycloalkylalkyl groups areexemplified by cyclopropylmethyl, cyclohexylethyl and the like.

“Aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to12 carbon atoms inclusively having a single ring (e.g., phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl). Exemplary arylsinclude phenyl, pyridyl, naphthyl and the like.

“Arylalkyl” refers to an alkyl substituted with an aryl, i.e.,aryl-alkyl-groups, preferably having from 1 to 6 carbon atomsinclusively in the alkyl moiety and from 6 to 12 carbon atomsinclusively in the aryl moiety. Such arylalkyl groups are exemplified bybenzyl, phenethyl and the like.

“Heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” refer to alkyl,alkenyl and alkynyl as defined herein in which one or more of the carbonatoms are each independently replaced with the same or differentheteroatoms or heteroatomic groups. Heteroatoms and/or heteroatomicgroups which can replace the carbon atoms include, but are not limitedto, —O—, —S—, —S—O—, —NR^(γ)—, —PH—, —S(O)—, —S(O)₂—, —S(O)NR^(γ)—,—S(O)₂NR^(γ)—, and the like, including combinations thereof, where eachR^(γ) is independently selected from hydrogen, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

“Heteroaryl” refers to an aromatic heterocyclic group of from 1 to 10carbon atoms inclusively and 1 to 4 heteroatoms inclusively selectedfrom oxygen, nitrogen and sulfur within the ring. Such heteroaryl groupscan have a single ring (e.g., pyridyl or furyl) or multiple condensedrings (e.g., indolizinyl or benzothienyl).

“Heteroarylalkyl” refers to an alkyl substituted with a heteroaryl,i.e., heteroaryl-alkyl-groups, preferably having from 1 to 6 carbonatoms inclusively in the alkyl moiety and from 5 to 12 ring atomsinclusively in the heteroaryl moiety. Such heteroarylalkyl groups areexemplified by pyridylmethyl and the like.

“Heterocycle”, “heterocyclic” and interchangeably “heterocycloalkyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, from 2 to 10 carbon ring atoms inclusively andfrom 1 to 4 hetero ring atoms inclusively selected from nitrogen, sulfuror oxygen within the ring. Such heterocyclic groups can have a singlering (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings(e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples ofheterocycles include, but are not limited to, furan, thiophene,thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline,piperidine, piperazine, pyrrolidine, indoline and the like.

“Heterocycloalkylalkyl” refers to an alkyl substituted with aheterocycloalkyl, i.e., heterocycloalkyl-alkyl-groups, preferably havingfrom 1 to 6 carbon atoms inclusively in the alkyl moiety and from 3 to12 ring atoms inclusively in the heterocycloalkyl moiety.

“Oxy” refers to a divalent group —O—, which may have varioussubstituents to form different oxy groups, including ethers and esters.

“Alkoxy” or “alkyloxy” are used interchangeably herein to refer to thegroup —OR, wherein R is an alkyl group, including optionally substitutedalkyl groups.

“Aryloxy” as used herein refer to the group —OR wherein R is an arylgroup as defined above including optionally substituted aryl groups asalso defined herein.

“Carboxy” refers to —COOH.

“Carboxyalkyl” refers to an alkyl substituted with a carboxy group.

“Carbonyl” refers to the group —C(O)—. Substituted carbonyl refers tothe group R^(η)—C(O)—R^(η), where each R^(η) is independently selectedfrom optionally substituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy,carboxy, aryl, aryloxy, heteroaryl, heteroarylalkyl, acyl,alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typicalsubstituted carbonyl groups including acids, ketones, aldehydes, amides,esters, acyl halides, thioesters, and the like.

“Amino” refers to the group —NH₂. Substituted amino refers to the group—NHR^(η), NR^(η)R^(η), and NR^(η)R^(η)R^(η), where each R^(η) isindependently selected from optionally substituted alkyl, cycloalkyl,cycloheteroalkyl, alkoxy, carboxy, aryl, aryloxy, heteroaryl,heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, andthe like. Typical amino groups include, but are limited to,dimethylamino, diethylamino, trimethylammonium, triethylammonium,methylysulfonylamino, furanyl-oxy-sulfamino, and the like.

“Aminoalkyl” refers to an alkyl group in which one or more of thehydrogen atoms are replaced with an amino group, including a substitutedamino group.

“Aminocarbonyl” refers to a carbonyl group substituted with an aminogroup, including a substituted amino group, as defined herein, andincludes amides.

“Aminocarbonylalkyl” refers to an alkyl substituted with anaminocarbonyl group, as defined herein.

“Halogen” or “halo” refers to fluoro, chloro, bromo and iodo.

“Haloalkyl” refers to an alkyl group in which one or more of thehydrogen atoms are replaced with a halogen. Thus, the term “haloalkyl”is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. upto perhaloalkyls. For example, the expression “(C₁ C₂) haloalkyl”includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl,1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1 trifluoroethyl,perfluoroethyl, etc.

“Hydroxy” refers to —OH.

“Hydroxyalkyl” refers to an alkyl substituted with one or more hydroxygroup.

“Thio” or “sulfanyl” refers to —SH. Substituted thio or sulfanyl refersto —S—R^(η), where R^(η) is an alkyl, aryl or other suitablesubstituent.

“Alkylthio” refers to —SR, where R is an alkyl, which can be optionallysubstituted. Typical alkylthio group include, but are not limited to,methylthio, ethylthio, n-propylthio, and the like.

“Alkylthioalkyl” refers to an alkyl substituted with an alkylthio group,—SR, where R is an alkyl, which can be optionally substituted.

“Sulfonyl” refers to —SO₂—. Substituted sulfonyl refers to —SO₂—R^(η),where R^(η) is an alkyl, aryl or other suitable substituent.

“Alkylsulfonyl” refers to —SO₂—R^(ζ), where R^(ζ) is an alkyl, which canbe optionally substituted. Typical alkylsulfonyl groups include, but arenot limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and thelike.

“Alkylsulfonylalkyl” refers to an alkyl substituted with analkylsulfonyl group, —SO₂—R^(ζ), where R^(ζ) is an alkyl, which can beoptionally substituted.

“Membered ring” is meant to embrace any cyclic structure. The numberpreceding the term “membered” denotes the number of skeletal atoms thatconstitute the ring. Thus, for example, cyclohexyl, pyridine, pyran andthiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, andthiophene are 5-membered rings.

“Fused bicyclic ring” refers to both unsubstituted and substitutedcarbocyclic and/or heterocyclic ring moieties having 5 or 8 atoms ineach ring, the rings having 2 common atoms.

“Optionally substituted” as used herein with respect to the foregoingchemical groups means that positions of the chemical group occupied byhydrogen can be substituted with another atom, such as carbon, oxygen,nitrogen, or sulfur, or a chemical group, exemplified by, but notlimited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substitutedalkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo,methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substitutedalkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio,alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substitutedcarboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino,sulfonamido, substituted sulfonamido, cyano, amino, substituted amino,alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo,hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl,arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substitutedheteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl,cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle,(heterocycle)oxy, and (heterocycle)alkyl; where preferred heteroatomsare oxygen, nitrogen, and sulfur. Additionally, where open valencesexist on these substitute chemical groups they can be furthersubstituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocyclegroups, that where these open valences exist on carbon they can befurther substituted by halogen and by oxygen-, nitrogen-, orsulfur-bonded substituents, and where multiple such open valences exist,these groups can be joined to form a ring, either by direct formation ofa bond or by formation of bonds to a new heteroatom, preferably oxygen,nitrogen, or sulfur. It is further contemplated that the abovesubstitutions can be made provided that replacing the hydrogen with thesubstituent does not introduce unacceptable instability to the moleculesof the present invention, and is otherwise chemically reasonable. One ofordinary skill in the art would understand that with respect to anychemical group described as optionally substituted, only stericallypractical and/or synthetically feasible chemical groups are meant to beincluded. Finally, “optionally substituted” as used herein refers to allsubsequent modifiers in a term or series of chemical groups. Forexample, in the term “optionally substituted arylalkyl,” the “alkyl”portion and the “aryl” portion of the molecule may or may not besubstituted, and for the series “optionally substituted alkyl,cycloalkyl, aryl and heteroaryl,” the alkyl, cycloalkyl, aryl, andheteroaryl groups, independently of the others, may or may not besubstituted.

6.3 Engineered Imine Reductase (IRED) Polypeptides

The present invention provides engineered polypeptides having iminereductase activity, polynucleotides encoding the polypeptides; methodsof preparing the polypeptides, and methods for using the polypeptides.Where the description relates to polypeptides, it is to be understoodthat it also describes the polynucleotides encoding the polypeptides.

As noted above, imine reductases belong to a class of enzymes thatcatalyze the reductive amination of a ketone substrate and a primary orsecondary amine substrate to a secondary or tertiary amine product, asillustrated by Scheme 1 (see above for Scheme and group structures forcompounds of formula (I), (II), and (III)).

The opine dehydrogenase from Arthrobacter sp. strain 1C (also referredto herein as “CENDH”) having the amino acid sequence of SEQ ID NO:2,naturally catalyzes the conversion of ketone substrate, pyruvate and theamino acid substrate, L-2-amino pentanoic acid (or “L-norvaline”) to theproduct (2S)-2-((1-carboxyethyl)amino)pentanoic acid. CENDH alsocatalyzes the reaction of pyruvate with the amino acid substrates,L-ornithine, and β-alanine, and structurally similar amino sulfonic acidsubstrate, taurine. In addition, CENDH was found to catalyze theconversion of the unactivated ketone substrate, cyclohexanone (ratherthan pyruvate) and its natural amine substrate, L-norvaline, to thesecondary amine product, (S)-2-(cyclohexylamino)pentanoic acid. CENDHalso was found to catalyze the conversion of its natural ketonesubstrate pyruvate with the primary amines butylamine, ethylamine, andisopropylamine, to their respective 2-(alkylamino)propanoic acidsecondary amine products. CENDH, however, did not exhibit any activityfor the conversion of pyruvate with secondary amines, such asdimethylamine. Furthermore, CENDH did not show any imine reductaseactivity with the unactivated ketone substrate, cyclohexanone, when usedtogether with the unactivated primary amine substrate, butylamine.

The present invention provides engineered imine reductases that overcomethe deficiencies of the wild-type opine dehydrogenase CENDH. Theengineered imine reductase polypeptides derived from the wild-typeenzyme of Arthrobacter sp. strain 1C are capable of efficientlyconverting pyruvate and L-norvaline to the product(2S)-2-((1-carboxyethyl)amino)pentanoic acid, but also capable ofefficiently converting a range of ketone substrate compounds of formula(I) and amine substrate compounds of formula (II), to the secondary andtertiary amine product compounds of formula (III) as shown by conversionreactions (a) through (s) which are listed below in Table 2.

TABLE 2 Conversion Reactions Conversion Substrate Reaction SubstrateCompound of Compound of Product Compound(s) of ID Formula (I) Formula(II) Formula (III) (a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

(m)

(n)

(o)

(p)

(q)

(r)

(s)

Significantly, the present invention provides amino acid residuepositions and corresponding mutations in the sequence of the referenceengineered polypeptide having imine reductase activity of SEQ ID NO:6(which was previously evolved from the naturally occurring CENDHpolypeptide of SEQ ID NO:2) that result in improved enzyme properties,including among others, imine reductase activity, substrate specificity,selectivity, thermal stability and solvent stability. In particular, thepresent invention provides engineered WED polypeptides capable ofcatalyzing reductive amination reactions such as those of Table 2 (i.e.,the reductive amination of ketone substrate compounds of formula (I)(e.g., cyclohexanone) with primary and secondary amine substratecompounds of formula (II) thereby producing secondary or tertiary aminecompounds of formula (III)).

In some embodiments, the engineered imine reductase polypeptides show anincreased activity in the conversion of the ketone substrate of formula(I) and amine substrate of formula (II) to an amine product of formula(III), in a defined time with the same amount of enzyme as compared tothe wild-type enzyme, CENDH. In some embodiments, the engineered iminereductase polypeptide has at least about 1.2 fold, 1.5 fold, 2 fold, 3fold, 4 fold, 5 fold, or 10 fold or more the activity as compared to thereference engineered polypeptide of SEQ ID NO:6 and/or SEQ ID NO:12,under suitable reaction conditions.

In some embodiments, the engineered imine reductase polypeptides exhibitan imine reductase activity in the conversion of a ketone substrate offormula (I) and an amine substrate of formula (II) to an amine productof formula (III), for which the wild-type polypeptide of SEQ ID NO:2,CENDH, has no detectable activity.

The product compounds of formula (III) produced by the engineered iminereductase polypeptides can be a secondary or tertiary amine compoundshaving one or more chiral centers. In some embodiments, the engineeredimine reductase polypeptides are capable of converting the ketone andamine substrate compounds of formula (I) and formula (II), to a chiralamine product compound of formula (III), in an enantiomeric excess ordiastereomeric excess of greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or greater.

In some embodiments, the engineered imine reductase polypeptides arecapable of converting the ketone and amine substrate compounds offormula (I) and formula (II) with increased tolerance for the presenceof one or both of these substrate compounds relative to the tolerance ofthe reference polypeptide of SEQ ID NO:6 and/or SEQ ID NO:12, undersuitable reaction conditions. Thus, in some embodiments the engineeredimine reductase polypeptides are capable of converting the ketone andamine substrate compounds of formula (I) and formula (II) at a substrateloading concentration of at least about 10 g/L, about 20 g/L, about 30g/L, about 40 g/L, about 50 g/L, about 70 g/L, about 100 g/L, about 125g/L, about 150 g/L. about 175 g/L or about 200 g/L or more with apercent conversion of at least about 40%, at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, at least about 98%, or at least about 99%, in areaction time of about 120 h or less, 72 h or less, about 48 h or less,about 36 h or less, or about 24 h less, under suitable reactionconditions.

The suitable reaction conditions under which the above-describedimproved properties of the engineered polypeptides carry out theconversion can be determined with respect to concentrations or amountsof polypeptide, substrate, cofactor (e.g., NAD(P)H), coenzyme (e.g., FDHor GDH), buffer, co-solvent, pH, temperature, reaction time, and/orconditions with the polypeptide immobilized on a solid support, asfurther described below and in the Examples.

The present invention provides numerous exemplary engineeredpolypeptides having imine reductase activity. These exemplarypolypeptides were evolved from the previously engineered polypeptide ofSEQ ID NO:6 (which was derived via directed evolution from the wild-typeCENDH of SEQ ID NO:2) and exhibit improved properties, particularlyincreased activity and stability in the conversion of various ketone andamine substrates, including the conversion of compounds (1j) and (2b) tothe amine product compound (3o), the conversion of compounds (1j) and(2c) to the amine product compound (3p), the conversion of compounds(1j) and (2g) to the amine product compound (3q), the conversion ofcompounds (1i) and (2h) to the amine product compound (3r), and theconversion of compounds (1e) and (2d) to the amine product compound(3s). These exemplary engineered polypeptides having imine reductaseactivity have amino acid sequences (provided in the accompanyingSequence Listing as even-numbered sequence identifiers of SEQ IDNOS:8-924) that include one or more residue differences as compared toSEQ ID NO:6 at the following residue positions: X12, X18, X20, X26, X27,X29, X37, X57, X65, X74, X82, X87, X93, X94, X96, X108, X111, X126,X138, X140, X141, X142, X143, X153, X154, X156, X157, X158, X159, X163,X170, X175, X177, X195, X197, X200, X201, X220, X221, X223, X234, X241,X242, X253, X254, X256, X257, X259, X260, X261, X262, X263, X264, X265,X267, X270, X272, X273, X274, X276, X277, X278, X279, X281, X282, X283,X284, X291, X292, X295, X296, X326, and X352. The specific amino aciddifferences at each of these positions that are associated with theimproved properties of the exemplary polypeptides of Tables 3A-3Linclude: X12M, X18G, X20V, X26M/V, X27S, X29K, X37P, X57D/L/V, X65I/V,X74W, X82C/P/T, X87A, X93G/Y, X94N, X96C, X108S, X111A/H, X126S, X138L,X140M, X141M/N/W, X142A, X143F/L/W/Y, X153E/F/Y,X154C/D/F/GM/L/N/Q/S/TN/Y, X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V,X159C/L/Q/V, X163V, X170F/K/R/S, X175R, X177R, X195S, X197V, X200S,X201I, X220C/K/Q, X221F, X223S, X234V/C/L, X241K, X242C/L, X253K/N,X254R, X256A/E/I/L/S/T/V, X257Q, X259C/I/L/M/R/T, X260A/D/G/N/Q/V/Y,X261E/F/H/L/P/Q/R/Y, X262F/G/P/V, X263C/D/E/H/I/K/L/M/N/P/Q/V, X264V,X265L, X267E/G/H/I/N/S, X270L, X272D, X273C/W, X274L/M/S, X276L,X277A/H/1/L, X278E/HM/N/R/S/W, X279L/T, X281A, X282A/R, X283M/V,X284C/F/H/L/P/Q/S, X291E, X292E/P, X295F, X296N, X326V, and X352Q. Inparticular, the amino acid residue differences X12M, X82C/P/T, andX111A/H, are associated with increased imine reductase activity and/orstability across a range of ketone and amine substrates (as shown byresults in Tables 3A-3L).

The structure and function information for exemplary non-naturallyoccurring (or engineered) imine reductase polypeptides of the presentinvention are based on five different high-throughput (HTP) screeningassays used in the directed evolution of these enzymes: (1) theconversion of the ketone and amine substrate compounds (1j) and (2b) tothe amine product compound (3o); (2) the conversion of the ketone andamine substrate compounds (1j) and (2c) to the amine product compound(3p); (3) the conversion of the ketone and amine substrate compounds(1j) and (2g) to the amine product compound (3q); (4) the conversion ofthe ketone and amine substrate compounds (1i) and (2h) to the amineproduct compound (3r); and (5) the conversion of the ketone and aminesubstrate compounds (1e) and (2d) to the amine product compound (3s).The results of these HTP screening assays which are shown below inTables 3A-3L. The odd numbered sequence identifiers (i.e., SEQ ID NOs)refer to the nucleotide sequence encoding the amino acid sequenceprovided by the even numbered SEQ ID NOs, and the sequences are providedin the electronic sequence listing file accompanying this invention,which is hereby incorporated by reference herein. The amino acid residuedifferences listed in Table 3A are based on comparison to the referencesequence of SEQ ID NO:6, which is the amino acid sequence of anengineered polypeptide having the following 29 residue differences ascompared to the opine dehydrogenase from Arthrobacter sp. strain 1C,CENDH: S29R, N94K, A111R, S137N, K156T, G157L, V184Q, V197I, N198E,M201L, Q220H, L223T, S232A, H259V, E261I, S266T, A279V, Y280L, A284M,I287T, N288S, R292V, Y293H, F295S, A311V, D324L, S328E, T332V, andG353E. The amino acid residue differences listed in Tables 3B-3L, arebased on comparison to the amino acid sequence of an engineeredpolypeptide of SEQ ID NO:12 which (as shown in Table 3A) has thefollowing 9 residue differences as compared to the reference sequence ofSEQ ID NO:6: R111A, T141W, N153Y, A154F, C256E, V274M, I283V, M284S, andE296N.

The activity of the engineered imine reductase polypeptides wasdetermined relative to the activity of a reference (or control)engineered polypeptide (as cited in the Table) using one or more of thefollowing five high-throughput (HTP) assays as the primary screen: (1)the conversion of the ketone and amine substrate compounds (1j) and (2b)to the amine product compound (3o); (2) the conversion of the ketone andamine substrate compounds (1j) and (2c) to the amine product compound(3p); (3) the conversion of the ketone and amine substrate compounds(1j) and (2g) to the amine product compound (3q); (4) the conversion ofthe ketone and amine substrate compounds (1i) and (2h) to the amineproduct compound (3r); and (5) the conversion of the ketone and aminesubstrate compounds (1e) and (2d) to the amine product compound (3s).The HTP assay values were determined using E. coli clear cell lysates in96 well-plate format of ˜100 μL, volume per well following assayreaction conditions as noted in the Tables.

TABLE 3A Engineered Polypeptides and Relative Enzyme Improvements SEQIncreased Activity¹ ID (1j) + (2b) → (3o) NO: Amino Acid DifferencesAssay² (nt/aa) (Relative to SEQ ID NO: 6) 44° C., 15% DMSO  7/8 R111A;R143W; N153Y; C256E; A273W; M284S; E296N; ++  9/10 R111A; N153Y; C256E;V274M; I283V; ++ 11/12 R111A; T141W; N153Y; A154F; C256E; V274M; I283V;+++ M284S; E296N; 13/14 R111A; T141W; N153Y; A273C; V274M; I283V; M284S;+++ 15/16 R111H; T141W; N153Y; A154F; A273W; V274L; I283V; +++ M284S;E296N; 17/18 N153Y; C256E; V274L; ++ 19/20 R111H; T141W; N153Y; A273W;V274M; M284S; E296N; +++ 21/22 V82P; R111A; T141W; N153Y; V274M; I283V;M284S; ++ 23/24 T141W; N153Y; I283V; +++ 25/26 V82P; T141W; N153Y;C256E; V274M; I283V; +++ 27/28 N153Y; ++ 29/30 R111A; N153Y; V274M;I283V; M284S; ++ 31/32 R111A; T141W; N153Y; A273C; I283V; +++ 33/34V82P; R111A; T141W; N153Y; C256E; V274M; I283V; +++ M284S; E296N; 35/36R111A; T141W; N153Y; C256E; A273W; V274L; I283V; +++ M284S; E296V; 37/38R111H; T141W; N153Y; C256E; I283V; ++ 39/40 N153Y; A154F; C256E; V274M;M284S; + 41/42 T141W; N153Y; A154F; C256E; +++ 43/44 R111A; N153Y;C256E; I283V; E296V; ++ 45/46 N153Y; I283V; ++ 47/48 A93G; R143W; M159L;N277I; I326V; + 49/50 A93Y; A96C; V259M; V279L; + 51/52 T141W; C142A;M159L; C163V; V259R; ++ 53/54 A93Y; A96C; C142A; R143W; M159L; V259W;V279L; I326V; ++ 55/56 R143W; M159L; C163V; V259M; N277I; + 57/58 T141W;V259M; N277A; ++ 59/60 A93Y; C142A; M159L; C163V; V259M; V279L; ++ 61/62K94N; C142A; M159L; C163V; V259M; V279L; + 63/64 A93Y; C142A; R143W;C163V; V259M; V279L; ++ 65/66 R143W; V259W; V279L; ++ 67/68 R143W;M159L; V259M; N277I; V279L; ++ 69/70 A93Y; K94N; R143W; M159L; V259M;V279L; ++ 71/72 K94N; R143W; M159L; C163V; V259M; V279L; +++ 73/74 K94N;T141W; M159L; V259M; N277A; I326V; + 75/76 K94N; C142A; R143W; M159L;V259M; I326V; ++ ¹Levels of increased activity were determined relativeto the reference polypeptide of SEQ ID NO: 6 and defined as follows: “−”= activity less than or equal to reference polypeptide; “+” = at least2-fold but less than 4-fold increased activity; “++” = at least 4-foldbut less than 8-fold increased activity; “+++” = at least 8-foldincreased activity but less than 16-fold. ²Substrate Compounds (1j) and(2b) Conversion to Product Compound (3o) Activity Assay: Enzyme LysatePreparation: E. coli cells expressing the polypeptide variant gene ofinterest were pelleted, placed in 96-well plates and lysed in 250 μLlysis buffer (1 g/L lysozyme and 0.5 g/L PMBS in 0.1M phosphate buffer,pH 8.0) with low-speed shaking for 2 h on titre-plate shaker at roomtemperature. The lysate containing plates were centrifuged at 4000 rpmand 4° C. for 20 min and the clear lysate supernatant used for assayreactions. HTP Assay Reaction: The enzyme assay reaction was carried outin a total volume of 100 μL in a 96-well plate format. The assayreaction was initiated by adding the following to each well containing45 μL of the lysate: (i) 20 μL of GDH cofactor recycling pre-mix(pre-mix contains 90 g/L glucose, 15 g/L NAD+, 5 g/L GDH-105); (ii) 20μL of (2b) stock solution (0.5 mM); and 15 μL ketone substrate (1j)stock solution (333 mM compound (1j) in DMSO). The resulting assayreaction included 50 mM ketone substrate compound (1j), 100 mM aminesubstrate (compound (2b)), 100 mM glucose, 3 g/L NAD+, 1 g/L GDH-105,100 mM potassium phosphate, pH 8.0, 15% (v/v) DMSO. The reaction platewas heat-sealed and shaken at 4000 rpm overnight (16-24 h) at 44° C.HPLC Work-up and Analysis: Each reaction mixture was quenched by adding100 μL CH₃CN with 0.1% formic acid, shaken, and centrifuged at 4000 rpmand 4° C. for 10 min. 20 μL of the quenched mixture was diluted 5-foldin 80 μL CH₃CN/H₂O (50/50) with 0.05% formic acid with mixing. 10 μLThese mixtures then were analyzed for product compound (3o) formation byHPLC as described in Example 3.

TABLE 3B Engineered Polypeptides and Relative Enzyme ImprovementsIncreased Increased Increased Increased Amino Acid Activity¹ Activity¹Activity¹ Activity¹ Differences (1j) + (2g) → (1i) + (2h) → (1e) + (2d)→ (1j) + (2c) → SEQ ID (Relative (3q) Assay² (3r) Assay³ (3s) Assay⁴(3p) Assay⁵ NO: to SEQ ID 44° C., pH 8.0, 44° C., pH 8.0, 44° C., pH8.0, 44° C., pH 8.0, (nt/aa) NO: 12) 15% DMSO 15% DMSO 30% DMSO 15% DMSO77/78 C142A; 1.25 n.d. n.d. n.d. M159L; V259M; 79/80 N108S; n.d. 1.061.00 1.26 81/82 F154Q; n.d. 1.84 0.36 1.51 83/84 V259L; 0.80 3.27 1.121.14 85/86 P267G; n.d. 3.00 0.75 0.76 87/88 T156L; n.d. 1.48 0.61 1.3289/90 V259T; n.d. 1.04 1.20 0.83 91/92 F154S; 3.32 3.37 0.69 3.08 93/94K260G; 1.15 1.92 1.04 1.27 95/96 P278H; 0.69 1.35 1.33 0.90 97/98 I242L;0.85 1.26 1.14 1.07 99/100 M274S; 0.27 0.28 0.28 1.34 101/102 A234L;1.11 1.18 0.79 1.33 103/104 F154G; 2.28 2.47 0.42 1.76 105/106 V259W;0.23 2.04 0.28 0.36 107/108 Y263C; 0.94 1.36 1.62 1.09 109/110 E256T;0.89 0.71 1.68 0.93 111/112 V82T; 2.05 1.55 1.73 1.52 113/114 T156H;1.73 1.10 1.05 2.26 115/116 Y263K; 1.93 1.11 0.52 1.56 117/118 Y263Q;1.63 1.05 1.00 1.31 119/120 P267S; 0.39 1.63 0.59 0.72 121/122 F154L;0.56 1.23 0.35 0.83 123/124 S284P; 1.32 1.05 0.65 1.30 125/126 F154N;1.67 2.15 1.57 2.21 127/128 I261L; 1.19 1.03 1.25 1.21 129/130 Y263M;1.91 1.15 0.78 1.47 131/132 Y263L; 2.85 1.58 0.59 1.78 133/134 F154Y;0.65 0.93 1.03 1.23 135/136 K260A; 1.21 1.56 0.77 1.88 137/138 W141M;0.10 1.49 0.55 0.45 139/140 Y263I; 1.46 1.62 0.69 1.10 141/142 K260D;0.79 1.29 0.64 1.59 143/144 T156M; 1.87 1.48 0.73 1.51 145/146 T156N;3.16 1.78 0.51 2.90 147/148 N277L; 0.46 0.51 1.37 0.49 149/150 V259C;0.66 0.45 1.23 0.86 151/152 P267E; 0.26 1.88 0.23 0.76 153/154 Y263V;0.57 3.10 1.32 0.90 155/156 F154T; 1.08 1.93 0.70 1.79 157/158 T223S;0.39 2.21 0.46 0.76 159/160 P278E; 0.95 1.40 1.18 1.11 161/162 V259M;0.92 3.04 1.71 0.98 163/164 R281A; 1.14 1.50 1.27 1.22 165/166 W141N;0.35 2.20 1.17 0.79 167/168 I261H; 1.51 1.37 2.00 1.38 169/170 V292E;0.98 0.63 1.88 1.14 171/172 F154D; 2.69 2.16 0.19 2.90 173/174 M159C;1.06 1.38 0.65 1.46 175/176 Y263H; 1.75 1.35 0.43 1.57 177/178 T156R;1.07 0.62 0.10 1.32 179/180 F154K; 0.77 1.26 0.26 1.32 181/182 L12M;1.12 1.24 1.28 1.39 183/184 P267H; 0.44 1.55 0.74 0.72 ¹Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 12. “n.d.” = not determined. ²Substrate Compounds (1j) +(2g) → Product Compound (3q) Activity Assay: Enzyme Lysate Preparation:E. coli cells expressing the polypeptide variant gene of interest werepelleted, placed in 96-well plates and lysed in 400 μL lysis buffer (1g/L lysozyme and 0.5 g/L PMBS in 0.1M phosphate buffer, pH 8.0) withlow-speed shaking for 2 h on titre-plate shaker at room temperature. Thelysate containing plates were centrifuged at 4000 rpm and 4° C. for 20min and the clear lysate supernatant used for assay reactions. HTP AssayReaction: The enzyme assay reaction was carried out in a total volume of100 μL in a 96-well plate format. The assay reaction was initiated byadding the following to each well containing 45 μL of the lysate: (i) 20μL of GDH cofactor recycling pre-mix (pre-mix contains 50 g/L glucose,15 g/L NAD+, 5 g/L GDH-105); (ii) 20 μL of (2g) stock solution (0.5 mM);and 15 μL ketone substrate (1j) stock solution (333 mM compound (1j) inDMSO). The resulting assay reaction included 50 mM ketone substratecompound (1j), 100 mM amine substrate (compound (2g)), 55.5 mM glucose,3 g/L NAD+, 1 g/L GDH-105, 100 mM potassium phosphate, pH 8.0, 15% (v/v)DMSO. The reaction plate was heat-sealed and shaken at 4000 rpmovernight (16-24 h) at 44° C. HPLC Work-up and Analysis: Each reactionmixture was quenched by adding 100 μL CH₃CN with 0.1% formic acid,shaken, and centrifuged at 4000 rpm and 4° C. for 10 min. 20 μL of thequenched mixture was diluted 5-fold in 80 μL CH₃CN/H₂O (50/50) with0.05% formic acid with mixing. 10 μL of these mixtures then wereanalyzed for product compound (3q) formation by HPLC as described inExample 3. ³Substrate Compounds (1i) + (2h) → Product Compound (3r)Activity Assay: Enzyme Lysate Preparation: E. coli cells expressing thepolypeptide variant gene of interest were pelleted, placed in 96-wellplates and lysed in 250 μL lysis buffer (1 g/L lysozyme and 0.5 g/L PMBSin 0.1M phosphate buffer, pH 8.0) with low-speed shaking for 2 h ontitre-plate shaker at room temperature. The lysate containing plateswere centrifuged at 4000 rpm and 4° C. for 20 min and the clear lysatesupernatant used for assay reactions. HTP Assay Reaction: The enzymeassay reaction was carried out in a total volume of 100 μL in a 96-wellplate format. The assay reaction was initiated by adding the followingto each well containing 45 μL of the lysate: (i) 20 μL of GDH cofactorrecycling pre-mix (pre-mix contains 50 g/L glucose, 15 g/L NAD+, 5 g/LGDH-105); (ii) 20 μL of propylamine stock solution (500 mM); and 15 μLketone substrate stock solution (333 mM compound (1i) in DMSO). Theresulting assay reaction included 50 mM ketone substrate compound (1i),100 mM amine substrate propylamine (compound (2h)), 55.5 mM glucose, 3g/L NAD+, 1 g/L GDH-105, 100 mM potassium phosphate, pH 8.0, 15% (v/v)DMSO. The reaction plate was heat-sealed and shaken at 4000 rpmovernight (16-24 h) at 44° C. LC-MS Work-up and Analysis: Each reactionmixture was quenched by adding 100 μL CH₃CN with 0.1% formic acid,shaken, and centrifuged at 4000 rpm and 4° C. for 10 min. 10 μL of thequenched mixture was diluted 20-fold in 190 μL CH₃CN/H₂O (50/50) with0.05% formic acid with mixing. 10 μL of this 20-fold dilution mixturewas then further diluted in 190 μL CH₃CN/H₂O (50/50) with 0.05% formicacid for a total 800 fold diluted mixtures. These mixtures then wereanalyzed for product compound (3r) formation by LC-MS in MRM mode asdescribed in Example 3. ⁴Substrate Compounds (1e) + (2d) → ProductCompound (3s) Activity Assay: Enzyme Lysate Preparation: E. coli cellsexpressing the polypeptide variant gene of interest were pelleted,placed in 96-well plates and lysed in 250 μL lysis buffer (1 g/Llysozyme and 0.5 g/L PMBS in 0.1M phosphate buffer, pH 8.0) withlow-speed shaking for 2 h on titre-plate shaker at room temperature. Thelysate containing plates were centrifuged at 4000 rpm and 4° C. for 20min and the clear lysate supernatant used for assay reactions. HTP AssayReaction: The enzyme assay reaction was carried out in a total volume of100 μL in a 96-well plate format. The assay reaction was initiated byadding the following to each well containing 50 μL of the lysate: (i) 20μL of GDH cofactor recycling pre-mix (pre-mix contains 50 g/L glucose,15 g/L NAD+, 5 g/L GDH-105); (ii) 15 μL of aniline stock solution (667mM in DMSO); and 15 μL ketone substrate stock solution (333 mM compound(1e) in DMSO). The resulting assay reaction included 50 mM ketonesubstrate compound (1e), 100 mM amine substrate (compound (2d)), 55.5 mMglucose, 3 g/L NAD+, 1 g/L GDH-105, 100 mM potassium phosphate, pH 8.0,15% (v/v) or 30% (v/v) DMSO. The reaction plate was heat-sealed andshaken at 4000 rpm overnight (16-24 h) at 44° C. Work-up and Analysis:Each reaction mixture was quenched by adding 100 μL CH₃CN with 0.1%formic acid, shaken, and centrifuged at 4000 rpm and 4° C. for 10 min.10 μL of the quenched mixture was diluted 20-fold in 190 μL CH₃CN/H₂O(50/50) with 0.05% formic acid with mixing. 10 μL of this 20-folddilution mixture was then further diluted in 190 μL CH₃CN/H₂O (50/50)with 0.05% formic acid for a total 800 fold diluted mixtures. Thesemixtures then were analyzed for product compound (3s) formation by LC-MSin MRM mode as described in Example 3. ⁵Substrate Compounds (1j) + (2c)→ Product Compound (3p) Activity Assay: Enzyme Lysate Preparation: E.coli cells expressing the polypeptide variant gene of interest werepelleted, placed in 96-well plates and lysed in 400 μL lysis buffer (1g/L lysozyme and 0.5 g/L PMBS in 0.1M phosphate buffer, pH 8.0) withlow-speed shaking for 2 h on titre-plate shaker at room temperature. Thelysate containing plates were centrifuged at 4000 rpm and 4° C. for 20min and the clear lysate supernatant used for assay reactions. HTP AssayReaction: The enzyme assay reaction was carried out in a total volume of100 μL in a 96-well plate format. The assay reaction was initiated byadding the following to each well containing 45 μL of the lysate: (i) 20μL of GDH cofactor recycling pre-mix (pre-mix contains 50 g/L glucose,15 g/L NAD+, 5 g/L GDH-105); (ii) 20 μL of methylamine stock solution(0.5 mM); and 15 μL ketone substrate stock solution (333 mM compound(1j) in DMSO). The resulting assay reaction included 50 mM ketonesubstrate compound (1j), 100 mM amine substrate (compound (2c)), 55.5 mMglucose, 3 g/L NAD+, 1 g/L GDH-105, 100 mM potassium phosphate, pH 8.0,15% (v/v) DMSO. The reaction plate was heat-sealed and shaken at 4000rpm overnight (16-24 h) at 44° C. LC-MS Work-up and Analysis: Eachreaction mixture was quenched by adding 100 μL CH₃CN with 0.1% formicacid, shaken, and centrifuged at 4000 rpm and 4° C. for 10 min. 10 μL ofthe quenched mixture was diluted 20-fold in 190 μL CH₃CN/H₂O (50/50)with 0.05% formic acid with mixing. 10 μL of this 20-fold dilutionmixture was then further diluted in 190 μL CH₃CN/H₂O (50/50) with 0.05%formic acid for a total 800 fold diluted mixtures. These mixtures thenwere analyzed for product compound (3p) formation by either LC-MS in MRMas described in Example 3.

TABLE 3C Engineered Polypeptides and Relative Activity ImprovementsIncreased Activity¹ (1j) + (2g) → (3q) Assay² SEQ ID Amino AcidDifferences 44° C., pH 8.0, NO: (nt/aa) (Relative to SEQ ID NO: 12) 15%DMSO 197/198 A37P; V82T; T156N; Y263Q; ++++ 187/188 V82T; T156N; I261H;Y263Q; ++++ 189/190 A37P; T156N; I261H; Y263Q; +++ 195/196 A37P; T156N;Y263Q; +++ 185/186 A37P; T156N; Y263H; +++ 193/194 A37P; V82T; T156N;Y263H; ++ 191/192 A37P; V82T; T156N; I261H; ++ Y263H; ¹Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 146 and defined as follows: “+” = at least 1.2-fold butless than 3-fold increased activity; “++” = at least 3-fold but lessthan 4-fold increased activity; “+++” = at least 4-fold increasedactivity but less than 6-fold; and “++++“ = at least 6-fold increasedactivity but less than 8-fold. ²Substrate Compounds (1j) + (2g) →Product Compound (3q) Activity Assay: Enzyme Lysate Preparation: E. colicells expressing the polypeptide variant gene of interest were pelleted,placed in 96-well plates and lysed in 400 μL lysis buffer (1 g/Llysozyme and 0.5 g/L PMBS in 0.1M phosphate buffer, pH 8.0) withlow-speed shaking for 2 h on titre-plate shaker at room temperature. Thelysate containing plates were centrifuged at 4000 rpm and 4° C., for 20min and the clear lysate supernatant used for assay reactions. HTP AssayReaction: The enzyme assay reaction was carried out in a total volume of100 μL in a 96-well plate format. The assay reaction was initiated byadding the following to each well containing 45 μL of the lysate: (i) 20μL of GDH cofactor recycling pre-mix (pre-mix contains 50 g/L glucose,15 g/L NAD+, 5 g/L GDH-105); (ii) 20 μL of (2g) stock solution (0.5 mM);and 15 μL ketone substrate (1j) stock solution (333 mM compound (1j) inDMSO). The resulting assay reaction included 50 mM ketone substratecompound (1j), 100 mM amine substrate (compound (2g)), 55.5 mM glucose,3 g/L NAD+, 1 g/L GDH-105, 100 mM potassium phosphate, pH 8.0, 15% (v/v)DMSO. The reaction plate was heat-sealed and shaken at 4000 rpmovernight (16-24 h) at 44° C. HPLC Work-up and Analysis: Each reactionmixture was quenched by adding 100 μL CH₃CN with 0.1% formic acid,shaken, and centrifuged at 4000 rpm and 4° C. for 10 mM. 20 μL of thequenched mixture was diluted 5-fold in 80 μL CH₃CN/H₂O (50/50) with0.05% formic acid with mixing. 10 μL of these mixtures then wereanalyzed for product compound (3q) formation by HPLC as described inExample 3.

TABLE 3D Engineered Polypeptides and Relative Activity ImprovementsIncreased Activity¹ SEQ ID (1i) + (2h) → (3r) Assay² NO: Amino AcidDifferences 44° C., pH 8.0, (nt/aa) (Relative to SEQ ID NO: 12) 15% DMSO199/200 V82T; V259L; P267G; + 201/202 V82T; T2235; V259L; P267G; +++203/204 V82T; V259L; Y263L; P267S; ++ 205/206 V82T; T2235; V259L; P267E;++ R281A; 207/208 V82T; V259L; P267H; R281A; ++ 209/210 V82T; T156N;T2235; V259L; +++ 211/212 V82T; V259L; P267E; + 213/214 V82T; T156N;V259L; +++ 215/216 V82T; V259L; R281A; + 217/218 V82T; V259L; + 219/220V82T; V259L; P267E; R281A; ++ 221/222 V82T; T156N; V259L; R281A; +++223/224 V82T; T2235; V259L; + 225/226 T2235; V259L; + 227/228 V82T;T156N; T2235; V259L; ++++ P267G; R281A; 229/230 T2235; V259L; Y263V;P267S; ++ 231/232 T156N; V259L; R281A; + 233/234 F154N; T156N; V259L; +235/236 V82T; T2235; V259L; P267H; ++ ¹Levels of increased activity weredetermined relative to the reference polypeptide of SEQ ID NO: 84 anddefined as follows: “+” = at least 1.2-fold but less than 3-foldincreased activity; “++”, at least 3-fold but less than 4-fold increasedactivity; “+++” = at least 4-fold increased activity but less than10-fold; and “++++” = at least 10-fold increased activity but less than15-fold. ²Substrate Compounds (1i) + (2h) → Product Compound (3r)Activity Assay: Enzyme Lysate Preparation: E. coli cells expressing thepolypeptide variant gene of interest were pelleted, placed in 96-wellplates and lysed in 250 μL lysis buffer (1 g/L lysozyme and 0.5 g/L PMBSin 0.1M phosphate buffer, pH 8.0) with low-speed shaking for 2 h ontitre-plate shaker at room temperature. The lysate containing plateswere centrifuged at 4000 rpm and 4° C. for 20 min and the clear lysatesupernatant used for assay reactions. HTP Assay Reaction: The enzymeassay reaction was carried out in a total volume of 100 μL in a 96-wellplate format. The assay reaction was initiated by adding the followingto each well containing 45 μL of the lysate: (i) 20 μL of GDH cofactorrecycling pre-mix (pre-mix contains 50 g/L glucose, 15 g/L NAD+, 5 g/LGDH-105); (ii) 20 μL of propylamine stock solution (500 mM); and 15 μLketone substrate stock solution (333 mM compound (1i) in DMSO). Theresulting assay reaction included 50 mM ketone substrate (compound (1i),100 mM amine substrate propylamine compound (2h)), 55.5 mM glucose, 3g/L NAD+, 1 g/L GDH-105, 100 mM potassium phosphate, pH 8.0, 15% (v/v)DMSO. The reaction plate was heat-sealed and shaken at 4000 rpmovernight (16-24 h) at 44° C. LC-MS Work-up and Analysis: Each reactionmixture was quenched by adding 100 μL CH₃CN with 0.1% formic acid,shaken, and centrifuged at 4000 rpm and 4° C. for 10 min. 10 μL of thequenched mixture was diluted 20-fold in 190 μL CH₃CN/H₂O (50/50) with0.05% formic acid with mixing. 10 μL of this 20-fold dilution mixturewas then further diluted in 190 μL CH₃CN/H₂O (50/50) with 0.05% formicacid for a total 800 fold diluted mixtures. These mixtures then wereanalyzed for product compound (3r) formation by LC-MS in MRM mode asdescribed in Example 3.

TABLE 3E Engineered Polypeptides and Relative Activity ImprovementsIncreased Activity¹ SEQ ID (1j) + (2c) → (3p) Assay² NO: Amino AcidDifferences 44° C., pH 8.0, (nt/aa) (Relative to SEQ ID NO: 12) 15% DMSO237/238 L12M; F154D; M159C; K260D; + Y263E; 239/240 F154S; Y263E; +241/242 L12M; F154S; T156M; + 243/244 F154S; A234L; S262P; Y263E; +245/246 F154S; S262P; + 247/248 L12M; F154S; M159C; K260A; + S262P;Y263E; 249/250 F154N; T156M; Y263P; + 251/252 F154T; T156N; A234L; +253/254 F154S; I261H; Y263E; + 255/256 L12M; F154S; T156M; M159C; +A234L; 257/258 F154S; T156M; A234L; K260A; + 259/260 V82T; F154T;T156M; + 261/262 L12M; F154S; T156N; M159C; + Y263P; 263/264 F154S;I261P; Y263E; + 265/266 F154S; I261H; S262P; + 267/268 F154S; M159C;S262P; Y263E; + 269/270 F154S; T156M; K260D; ++ 271/272 L12M; F154S;Y263E; + 273/274 L12M; F154S; A234L; K260D; + I261P; S262P; 275/276F154S; T156N; K260D; I261P; ++ 277/278 V82T; F154S; K260A; I261H; +279/280 F154S; T156M; + 281/282 F154S; A234L; I261P; Y263E; ++ 283/284L12M; F154N; M159C; A234L; + K260D; S262P; 285/286 L12M; F154S; A234L;K260D; ++ I261H; Y263P; 287/288 L12M; F154S; M159C; Y263P; + 289/290V82T; F154D; T156M; I261P; ++++ 291/292 L12M; V82T; F154S; T156N; ++++I261P; S262P; Y263P; 293/294 V82T; F154S; M159C; A234L; + I261H; Y263E;295/296 F154N; T156M; Y263E; + 297/298 L12M; V82T; F154N; T156M; +++Y263E; 299/300 F154S; K260D; S262P; Y263E; + 301/302 L12M; F154T; T156H;Y263E; ++ 303/304 F154N; T156N; M159C; Y263E; ++ 305/306 F154N; T156N;M159C; A234L; + I261P; S262P; Y263E; 307/308 F154S; T156M; A234L; +309/310 F154S; S262P; Y263E; + 311/312 V82T; F154S; T156M; + 313/314F154S; M159C; K260D; I261P; + 315/316 L12M; F154S; I261P; + 317/318V82T; F154S; A234L; K260A; + Y263E; 319/320 V82T; F154S; T156H; M159C; +I261H; Y263E; 321/322 L12M; F154S; S262P; Y263E; + 323/324 F154S; A234L;S262P; + 325/326 F154S; K260D; S284P; ++ 327/328 F154S; S262P; Y263D; +329/330 F154S; M159C; I261P; S262P; + 331/332 F154S; T156M; M159C; +333/334 L12M; F154T; T156N; + 335/336 F154S; T156N; M159C; I261P; +S262P; 337/338 F154S; M159C; K260D; I261H; + S262P; Y263E; 339/340F154N; T156H; M159C; I261H; + S262P; 341/342 L12M; F154S; M159C;Y263E; + 343/344 F154S; M159C; A234L; I261H; + S262P; Y263P; 345/346L12M; F154S; Y263E; G264V; + 347/348 V82T; F154S; Y263E; ++ 349/350F154S; T156M; K260D; I261H; ++++ S262P; Y263E; 5284P; ¹Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 92 and defined as follows: “+” = at least 1.2-fold butless than 3-fold increased activity; “++” = at least 3-fold but lessthan 4-fold increased activity; “+++” = at least 4-fold increasedactivity but less than 6-fold; and “++++” = at least 6-fold increasedactivity but less than 8-fold. ²Substrate Compounds (1j) + (2c) →Product Compound (3p) Activity Assay: Enzyme Lysate Preparation: E. colicells expressing the polypeptide variant gene of interest were pelleted,placed in 96-well plates and lysed in 400 μL lysis buffer (1 g/Llysozyme and 0.5 g/L PMBS in 0.1M phosphate buffer, pH 8.0) withlow-speed shaking for 2 h on titre-plate shaker at room temperature. Thelysate containing plates were centrifuged at 4000 rpm and 4° C. for 20min and the clear lysate supernatant used for assay reactions. HTP AssayReaction: The enzyme assay reaction was carried out in a total volume of100 μL in a 96-well plate format. The assay reaction was initiated byadding the following to each well containing 45 μL of the lysate: (i) 20μL of GDH cofactor recycling pre-mix (pre-mix contains 50 g/L glucose,15 g/L NAD+, 5 g/L GDH-105); (ii) 20 μL of methylamine stock solution(0.5 mM); and 15 μL ketone substrate stock solution (333 mM compound(1j) in DMSO). The resulting assay reaction included 50 mM ketonesubstrate compound (1j), 100 mM amine substrate (compound (2c)), 55.5 mMglucose, 3 g/L NAD+, 1 g/L GDH-105, 100 mM potassium phosphate, pH 8.0,15% (v/v) DMSO. The reaction plate was heat-sealed and shaken at 4000rpm overnight (16-24 h) at 44° C. LC-MS Work-up and Analysis: Eachreaction mixture was quenched by adding 100 μL CH₃CN with 0.1% formicacid, shaken, and centrifuged at 4000 rpm and 4° C. for 10 min. 10 μL ofthe quenched mixture was diluted 20-fold in 190 μL CH₃CN/H₂O (50/50)with 0.05% formic acid with mixing. 10 μL of this 20-fold dilutionmixture was then further diluted in 190 μL CH₃CN/H₂O (50/50) with 0.05%formic acid for a total 800 fold diluted mixtures. These mixtures thenwere analyzed for product compound (3p) formation by either LC-MS in MRMas described in Example 3.

TABLE 3F Engineered Polypeptides and Relative Activity ImprovementsIncreased Activity¹ SEQ (1e) + (2d) → (3s) Assay² ID NO: Amino AcidDifferences 44° C., pH 8.0, (nt/aa) (Relative to SEQ ID NO: 12) 30% DMSO351/352 L12M; Y263C; N277L; + 353/354 L12M; I261H; Y263C; N277L; ++V292E; 355/356 L12M; V259M; Y263C; V292E; ++ 357/358 V259M; Y263C; +359/360 L12M; V259M; I261H; Y263C; + P278H; 361/362 L12M; V259L;Y263C; + 363/364 L12M; Y263C; V292E; + 365/366 L12M; V259L; I261H;Y263C; + N277L; V292E; 367/368 V259M; Y263C; P278H; V292E; + 369/370L12M; V259M; Y263C; + 371/372 L12M; V259L; Y263C; P278H; + V292E;373/374 L12M; V259M; I261H; Y263C; + P278H; V292E; 375/376 V259M; Y263C;V292E; + ¹Levels of increased activity were determined relative to thereference polypeptide of SEQ ID NO: 162 and defined as follows: “+” = atleast 1.2-fold but less than 3-fold increased activity; “++” = at least3-fold but less than 4-fold increased activity. ²Substrate Compounds(1e) + (2d) → Product Compound (3s) Activity Assay: Enzyme LysatePreparation: E. coli cells expressing the polypeptide variant gene ofinterest were pelleted, placed in 96-well plates and lysed in 250 μLlysis buffer (1 g/L lysozyme and 0.5 g/L PMBS in 0.1M phosphate buffer,pH 8.0) with low-speed shaking for 2 h on titre-plate shaker at roomtemperature. The lysate containing plates were centrifuged at 4000 rpmand 4° C. for 20 min and the clear lysate supernatant used for assayreactions. HTP Assay Reaction: The enzyme assay reaction was carried outin a total volume of 100 μL in a 96-well plate format. The assayreaction was initiated by adding the following to each well containing50 μL of the lysate: (i) 20 μL of GDH cofactor recycling pre-mix(pre-mix contains 50 g/L glucose, 15 g/L NAD+, 5 g/L GDH-105); (ii) 15μL of aniline stock solution (667 mM in DMSO); and 15 μL ketonesubstrate stock solution (333 mM compound (1e) in DMSO). The resultingassay reaction included 50 mM ketone substrate compound (1e), 100 mMamine substrate (compound (2d)), 55.5 mM glucose, 3 g/L NAD+, 1 g/LGDH-105, 100 mM potassium phosphate, pH 8.0, 15% (v/v) or 30% (v/v)DMSO. The reaction plate was heat-sealed and shaken at 4000 rpmovernight (16-24 h) at 44° C. Work-up and Analysis: Each reactionmixture was quenched by adding 100 μL CH₃CN with 0.1% formic acid,shaken, and centrifuged at 4000 rpm and 4° C. for 10 mM. 10 μL of thequenched mixture was diluted 20-fold in 190 μL CH₃CN/H₂O (50/50) with0.05% formic acid with mixing. 10 μL of this 20-fold dilution mixturewas then further diluted in 190 μL CH₃CN/H₂O (50/50) with 0.05% formicacid for a total 800 fold diluted mixtures. These mixtures then wereanalyzed for product compound (3s) formation by LC-MS in MRM mode asdescribed in Example 3.

TABLE 3G Engineered Polypeptides and Relative Activity ImprovementsIncreased Activity¹ Increased Activity¹ (1j) + (2g) → (3q) (1j) + (2g) →(3q) Assay² Assay³ SEQ ID Amino Acid Differences 44° C., pH 9.8, 30% 44°C., pH 8.0, 15% NO: (nt/aa) (Relative to SEQ ID NO: 12) DMSO DMSO377/378 A37P; V82T; T156N; A158T; Y263Q; + n.d. 379/380 A37P; V82T;T156N; A158S; Y263Q; + n.d. 381/382 A37P; V82T; T156N; A158L; Y263Q; +n.d. 383/384 A37P; V82T; T156N; Y263Q; + n.d. 385/386 A37P; V82T; T156N;G170F; G177R; + n.d. Y263Q; 387/388 A37P; V82T; T156N; G170K; Y263Q; ++n.d. 389/390 A37P; V82T; T156N; Q175R; Y263Q; + n.d. 391/392 R29K; A37P;V82T; T156N; Y263Q; + n.d. 393/394 A37P; V82T; T156N; L157Y; Y263Q; +n.d. 395/396 A37P; K74W; V82T; T156N; Y263Q; + n.d. 397/398 A37P; V82T;T156N; L157R; Y263Q; ++ n.d. 399/400 A37P; V82T; T156N; A158R; Y263Q; ++n.d. 401/402 A37P; A57L; V82T; T156N; Y263Q; ++ n.d. 403/404 A37P; A57V;V82T; T156N; Y263Q; ++ n.d. 405/406 K26M; A37P; V82T; T156N; Y263Q; ++n.d. 407/408 A37P; V82T; F154M; T156N; Y263Q; n.d. + 409/410 A37P; A57D;V82T; T156N; Y263Q; + n.d. 411/412 A37P; V82T; T156N; A158V; Y263Q; +++n.d. 413/414 A37P; V82T; T156N; G170S; Y263Q; + n.d. 415/416 G27S; A37P;V82T; T156N; Y263Q; + n.d. 417/418 K26V; A37P; V82T; T156N; Y263Q; +n.d. 419/420 A37P; V82T; G126S; T156N; Y263Q; + n.d. 421/422 A37P; V82T;T156N; Y263Q; A352Q; + n.d. 423/424 A37P; V82T; T156N; L157F; Y263Q; +n.d. 425/426 A37P; V82T; T156N; I261R; Y263Q; n.d. + 427/428 A37P; V82T;T156N; I261H; Y263Q; n.d. + 429/430 A37P; V82T; T156N; Y263Q; P267G;n.d. ++ 431/432 A37P; V82T; T156N; E256I; Y263Q; n.d. + 433/434 A37P;V82T; T156N; H220C; Y263Q; n.d. + 435/436 A37P; V82T; F154V; T156N;Y263Q; n.d. ++ 437/438 A37P; V82T; T156N; Y263Q; P267N; n.d. ++ 439/440A37P; V82T; T156N; V259I; Y263Q; n.d. +++ 441/442 A37P; V82T; M138L;T156N; Y263Q; n.d. + 443/444 A18G; A37P; V82T; T156N; Y263Q; n.d. ++445/446 A37P; V82T; T156N; V200S; Y263Q; n.d. + 447/448 A37P; V82T;T156N; Y263Q; P278S; n.d. + 449/450 A37P; V82T; T156N; Y263Q; I270L;n.d. + 451/452 A37P; V82T; T156N; S262F; Y263Q; n.d. + 453/454 A37P;V82T; T156N; Y263Q; P267S; n.d. ++ 455/456 A37P; V82T; T156N; R241K;Y263Q; n.d. + 457/458 A37P; V82T; F140M; T156N; Y263Q; n.d. + 459/460A37P; V82T; T156N; E256S; Y263Q; n.d. + 461/462 A37P; V82T; T156N;Y263Q; G282R; n.d. + 463/464 A37P; V82T; T156N; Y263Q; P267I; n.d. +465/466 A37P; V82T; T156N; Y263Q; P278W; n.d. + ¹Levels of increasedactivity were determined relative to the reference polypeptide of SEQ IDNO: 198 and defined as follows: “+” = at least 1.1-fold but less than1.5-fold increased activity; “++” = at least 1.5-fold but less than2-fold increased activity; “+++” = at least 2-fold increased activitybut less than 3-fold. “n.d.” = not determined. ²Substrate Compounds(1j) + (2g) → Product Compound (3q) Activity Assay (pH 9.8, 30% DMSO):Enzyme Lysate Preparation: E. coli cells expressing the polypeptidevariant gene of interest were pelleted, placed in 96-well plates andlysed in 250 μL lysis buffer (1 g/L lysozyme and 0.5 g/L PMBS in 0.1Mphosphate buffer, pH 8.0) with low-speed shaking for 2 h on titre-plateshaker at room temperature. The lysate containing plates werecentrifuged at 4000 rpm and 4° C. for 20 min and the clear lysatesupernatant used for assay reactions. HTP Assay Reaction: The enzymeassay reaction was carried out in a total volume of 100 μL in a 96-wellplate format. The assay reaction was initiated by adding the followingto each well containing 30 μL of the lysate: (i) 10 μL of GDH cofactorrecycling pre-mix (pre-mix contains 50 g/L glucose, 15 g/L NAD+, 5 g/LGDH-105); (ii) 20 μL of (2g) amine stock solution (0.5 mM amine pH 9.8);and 30 μL ketone substrate (1j) stock solution (167 mM compound (1j) inDMSO). (iii) 10 μL Bicarbonate buffer pH 9.8. The resulting assayreaction included 50 mM ketone substrate compound (1j), 100 mM aminesubstrate (compound (2g)), 55.5 mM glucose, 3 g/L NAD+, 1 g/L GDH-105,100 mM bicarbonate buffer, pH 9.8, 30% (v/v) DMSO. The reaction platewas heat-sealed and shaken at 4000 rpm overnight (16-24 h) at 44° C.HPLC Work-up and Analysis: Each reaction mixture was quenched by adding100 μL CH₃CN with 0.1% formic acid, shaken, and centrifuged at 4000 rpmand 4° C. for 10 min. 20 μL of the quenched mixture was diluted 5-foldin 80 μL CH₃CN/H₂O (50/50) with 0.05% formic acid with mixing. 10 μL ofthese mixtures then were analyzed for product compound (3q) formation byHPLC as described in Example 3. ³Substrate compounds (1j) + (2g) →product compound (3q) activity assay (pH 8.0, 15% DMSO): Enzyme LysatePreparation: E. coli cells expressing the polypeptide variant gene ofinterest were pelleted, placed in 96-well plates and lysed in 400 μLlysis buffer (1 g/L lysozyme and 0.5 g/L PMBS in 0.1M phosphate buffer,pH 8.0) with low-speed shaking for 2 h on titre-plate shaker at roomtemperature. The lysate containing plates were centrifuged at 4000 rpmand 4° C. for 20 min and the clear lysate supernatant used for assayreactions. HTP Assay Reaction: The enzyme assay reaction was carried outin a total volume of 100 μL in a 96-well plate format. The assayreaction was initiated by adding the following to each well containing45 μL of the lysate: (i) 20 μL of GDH cofactor recycling pre-mix(pre-mix contains 50 g/L glucose, 15 g/L NAD+, 5 g/L GDH-105); (ii) 20μL of (2g) stock solution (0.5 mM); and 15 μL ketone substrate (1j)stock solution (333 mM compound (1j) in DMSO). The resulting assayreaction included 50 mM ketone substrate compound (1j), 100 mM aminesubstrate (compound (2g)), 55.5 mM glucose, 3 g/L NAD+, 1 g/L GDH-105,100 mM potassium phosphate, pH 8.0, 15% (v/v) DMSO. The reaction platewas heat-sealed and shaken at 4000 rpm overnight (16-24 h) at 44° C.HPLC Work-up and Analysis: Each reaction mixture was quenched by adding100 μL CH₃CN with 0.1% formic acid, shaken, and centrifuged at 4000 rpmand 4° C. for 10 min. 20 μL of the quenched mixture was diluted 5-foldin 80 μL CH₃CN/H₂O (50/50) with 0.05% formic acid with mixing. 10 μL ofthese mixtures then were analyzed for product compound (3q) formation byHPLC as described in Example 3.

TABLE 3H Engineered Polypeptides and Relative Activity ImprovementsIncreased Activity¹ SEQ ID (1i) + (2h) → (3r) Assay² NO: Amino AcidDifferences 44° C., pH 8.0, 15% (nt/aa) (Relative to SEQ ID NO: 12) DMSO467/468 V82T; T156N; T195S; T223S; + V259L; P267G; R281A; 469/470 V82T;R143Y; T156N; T223S; + V259L; P267G; R281A; 471/472 V82T; T156N; I197V;T223S; + V259L; P267G; R281A; 473/474 V82T; R143F; T156N; T223S; +V259L; P267G; R281A; 475/476 V82T; H87A; T156N; T223S; + V259L; P267G;R281A; 477/478 V82T; T156N; T223S; V259L; + P267G; P278R; R281A; 479/480V82T; T156N; T223S; V259L; + P267G; V279T; R281A; 481/482 V82T; F154V;T156N; T223S; + V259L; P267G; R281A; 483/484 V82T; F154C; T156N;T223S; + V259L; P267G; R281A; 485/486 V82T; F154S; T156N; T223S; +V259L; P267G; R281A; 487/488 V82T; T156N; T223S; V259L; + S262G; P267G;R281A; 489/490 V82T; T156N; Y221F; T223S; + V259L; P267G; R281A; 491/492V82T; T156N; T223S; V259L; + P267G; P278K; R281A; 493/494 V82T; T156N;T223S; V259L; + Y263C; P267G; R281A; 495/496 V82T; T156N; T223S;V259L; + Y263N; P267G; R281A; 497/498 V82T; T156N; T223S; V259L; +I261F; P267G; R281A; 499/500 V82T; R143L; T156N; T223S; + V259L; P267G;R281A; 501/502 V82T; T156N; T223S; V259L; + P267G; P278H; R281A; 503/504V82T; T156N; T223S; V259L; + I261Y; P267G; R281A; ¹Levels of increasedactivity were determined relative to the reference polypeptide of SEQ IDNO: 228 and defined as follows: “+” = at least 1.1-fold but less than1.5-fold increased activity. ²Substrate compounds (1i) + (2h) → productcompound (3r) activity assay: Enzyme Lysate Preparation: E. coli cellsexpressing the polypeptide variant gene of interest were pelleted,placed in 96-well plates and lysed in 250 μL lysis buffer (1 g/Llysozyme and 0.5 g/L PMBS in 0.1M phosphate buffer, pH 8.0) withlow-speed shaking for 2 h on titre-plate shaker at room temperature. Thelysate containing plates were centrifuged at 4000 rpm and 4° C. for 20min and the clear lysate supernatant used for assay reactions. HTP AssayReaction: The enzyme assay reaction was carried out in a total volume of100 μL in a 96-well plate format. The assay reaction was initiated byadding the following to each well containing 45 μL of the lysate: (i) 20μL of GDH cofactor recycling pre-mix (pre-mix contains 50 g/L glucose,15 g/L NAD+, 5 g/L GDH-105); (ii) 20 μL of propylamine stock solution(500 mM); and 15 μL ketone substrate stock solution (333 mM compound(1i) in DMSO). The resulting assay reaction included 50 mM ketonesubstrate compound (1i), 100 mM amine substrate propylamine (compound(2h)), 55.5 mM glucose, 3 g/L NAD+, 1 g/L GDH-105, 100 mM potassiumphosphate, pH 8.0, 15% (v/v) DMSO. The reaction plate was heat-sealedand shaken at 4000 rpm overnight (16-24 h) at 44° C. HPLC Work-up andAnalysis: Each reaction mixture was quenched by adding 100 μL CH₃CN with0.1% formic acid, shaken, and centrifuged at 4000 rpm and 4° C. for 10min. 20 μL of the quenched mixture was diluted 5-fold in 80 μL CH₃CN/H₂O(50/50) with 0.05% formic acid with mixing. 10 μL of these mixtures thenwere analyzed for product compound (3r) formation by HPLC.

TABLE 3I Engineered Polypeptides and Relative Activity ImprovementsIncreased Activity¹ (1j) + (2c) → (3p) Assay² SEQ ID Amino AcidDifferences 44° C., pH 8.0, 15% NO: (nt/aa) (Relative to SEQ ID NO: 12)DMSO 505/506 F154S; T156M; K260D; I261H; + S262P; Y263E; S284P; S295F;507/508 F154S; T156M; K260D; I261H; + S262P; Y263E; P278N; S284P;509/510 F154S; T156M; K260D; I261H; + S262P; Y263E; N277H; S284P;511/512 V82T; F154S; T156M; K260D; + I261H; S262P; Y263E; S284P; 513/514F154S; T156M; K260D; I261H; + S262P; Y263E; G282A; S284P; 515/516 V82C;F154S; T156M; K260D; + I261H; S262P; Y263E; S284P; 517/518 F154S; T156M;L157R; K260D; + I261H; S262P; Y263E; S284P; 519/520 F154S; T156M; L157Q;K260D; + I261H; S262P; Y263E; S284P; 521/522 F154S; T156M; K260D;I261H; + S262P; Y263E; G276L; S284P; 523/524 F154S; T156M; L157T;K260D; + I261H; S262P; Y263E; S284P; 525/526 F154S; T156M; K260D;I261H; + S262P; Y263E; S284P; T291E; ¹Levels of increased activity weredetermined relative to the reference polypeptide of SEQ ID NO: 350 anddefined as follows: “+” = at least 1.1-fold but less than 2-foldincreased activity. ²Substrate Compounds (1i) + (2c) → Product Compound(3p) Activity Assay: Enzyme Lysate Preparation: E. coli cells expressingthe polypeptide variant gene of interest were pelleted, placed in96-well plates and lysed in 400 μL lysis buffer (1 g/L lysozyme and 0.5g/L PMBS in 0.1M phosphate buffer, pH 8.0) with low-speed shaking for 2h on titre-plate shaker at room temperature. The lysate containingplates were centrifuged at 4000 rpm and 4° C. for 20 min and the clearlysate supernatant used for assay reactions. HTP Assay Reaction: Theenzyme assay reaction was carried out in a total volume of 100 μL in a96-well plate format. The assay reaction was initiated by adding thefollowing to each well containing 45 μL of the lysate: (i) 20 μL of GDHcofactor recycling pre-mix (pre-mix contains 50 g/L glucose, 15 g/LNAD+, 5 g/L GDH-105); (ii) 20 μL of methylamine stock solution (0.5 mM);and 15 μL ketone substrate stock solution (333 mM compound (1j) inDMSO). The resulting assay reaction included 50 mM ketone substratecompound (1j), 100 mM amine substrate (compound (2c)), 55.5 mM glucose,3 g/L NAD+, 1 g/L GDH-105, 100 mM potassium phosphate, pH 8.0, 15% (v/v)DMSO. The reaction plate was heat-sealed and shaken at 4000 rpmovernight (16-24 h) at 44° C. HPLC Work-up and Analysis: Each reactionmixture was quenched by adding 100 μL CH₃CN with 0.1% formic acid,shaken, and centrifuged at 4000 rpm and 4° C. for 10 min. 20 μL of thequenched mixture was diluted 5-fold in 80 μL CH₃CN/H₂O (50/50) with0.05% formic acid with mixing. 10 μL of these mixtures then wereanalyzed for product compound (3p) formation by HPLC as described inExample 3.

TABLE 3J Engineered Polypeptides and Relative Activity ImprovementsIncreased Activity¹ SEQ (1e) + (2d) → (3s) Assay² ID NO: Amino AcidDifferences 44° C., pH 8.0, 30% (nt/aa) (Relative to SEQ ID NO: 12) DMSO527/528 L12M; M159V; I261H; Y263C; ++ N277L; V292E; 529/530 L12M; M159Q;I261H; Y263C; ++ N277L; V292E; 531/532 L12M; I261L; Y263C; N277L; ++V292E; 533/534 L12M; Y153F; I261H; Y263C; + N277L; V292E; 535/536 L12M;E256V; I261H; Y263C; ++ N277L; V292E; 537/538 L12M; K260H; I261H; Y263C;++ N277L; V292E; 539/540 L12M; I261H; Y263C; Q265L; ++ N277L; V292E;541/542 L12M; I261Q; Y263C; N277L; + V292E; 543/544 L12M; K260Q; I261H;Y263C; ++ N277L; V292E; 545/546 L12M; I242C; I261H; Y263C; ++ N277L;V292E; 547/548 L12M; S254R; I261H; Y263C; ++ N277L; V292E; 549/550 L12M;H220Q; I261H; Y263C; ++ N277L; V292E; 551/552 L12M; T156V; I261H;Y263C; + N277L; V292E; 553/554 L12M; A20V; I261H; Y263C; ++ N277L;V292E; 555/556 L12M; E256A; I261H; Y263C; + N277L; V292E; 557/558 L12M;E257Q; I261H; Y263C; +++ N277L; V292E; 559/560 L12M; K260Y; I261H;Y263C; +++ N277L; V292E; 561/562 L12M; E256L; I261H; Y263C; ++ N277L;V292E; 563/564 L12M; E2565; I261H; Y263C; ++ N277L; V292E; 565/566 L12M;L65I; I261H; Y263C; ++ N277L; V292E; 567/568 L12M; L201I; I261H; Y263C;++ N277L; V292E; 569/570 L12M; K260G; I261H; Y263C; ++ N277L; V292E;571/572 L12M; A234C; I261H; Y263C; +++ N277L; V292E; 573/574 L12M;P253K; I261H; Y263C; +++ N277L; V292E; 575/576 L12M; I261H; Y263C;N277L; ++ S284F; V292E; 577/578 L12M; I261R; Y263C; N277L; ++ V292E;579/580 L12M; L65V; I261H; Y263C; ++ N277L; V292E; 581/582 L12M; K260N;I261H; Y263C; + N277L; V292E; 583/584 L12M; V82T; I261H; Y263C; ++N277L; V292E; 585/586 L12M; I261H; Y263C; N277L; + S284H; V292E; 587/588L12M; I261H; Y263C; N277L; +++ S284L; V292E; 589/590 L12M; I261H; Y263C;N277L; +++ S284C; V292E; 591/592 L12M; H220K; I261H; Y263C; +++ N277L;V292E; 593/594 L12M; I261E; Y263C; N277L; ++ V292E; 595/596 L12M; I261H;Y263C; E272D; ++ N277L; V292E; 597/598 L12M; P253N; I261H; Y263C; ++N277L; V292E; 599/600 L12M; I261H; Y263C; N277L; +++ S284Q; V292E;601/602 L12M; K260V; I261H; Y263C; ++ N277L; V292E; 603/604 L12M; I261H;S262V; Y263C; +++ N277L; V292E; ¹Levels of increased activity weredetermined relative to the reference polypeptide of SEQ ID NO: 354 anddefined as follows: “+” = at least 1.1-fold but less than 1.5-foldincreased activity; “++” = at least 1.5-fold but less than 2-foldincreased activity; “+++” = at least 2-fold increased activity but lessthan 3-fold. ²Substrate Compounds (1e) + (2d) → Product Compound (3s)Activity Assay: Enzyme Lysate Preparation: E. coli cells expressing thepolypeptide variant gene of interest were pelleted, placed in 96-wellplates and lysed in 250 μL lysis buffer (1 g/L lysozyme and 0.5 g/L PMBSin 0.1M phosphate buffer, pH 8.0) with low-speed shaking for 2 h ontitre-plate shaker at room temperature. The lysate containing plateswere centrifuged at 4000 rpm and 4° C. for 20 min and the clear lysatesupernatant used for assay reactions. HTP Assay Reaction: The enzymeassay reaction was carried out in a total volume of 100 μL in a 96-wellplate format. The assay reaction was initiated by adding the followingto each well containing 50 μL of the lysate: (i) 20 μL of GDH cofactorrecycling pre-mix (pre-mix contains 50 g/L glucose, 15 g/L NAD+, 5 g/LGDH-105); (ii) 15 μL of aniline stock solution (667 mM in DMSO); and 15μL ketone substrate stock solution (333 mM compound (1e) in DMSO). Theresulting assay reaction included 50 mM ketone substrate compound (1e),100 mM amine substrate (compound (2d)), 55.5 mM glucose, 3 g/L NAD+, 1g/L GDH-105, 100 mM potassium phosphate, pH 8.0, 30% (v/v) DMSO. Thereaction plate was heat-sealed and shaken at 4000 rpm overnight (16-24h) at 44° C. Work-up and Analysis: Each reaction mixture was quenched byadding 100 μL CH₃CN with 0.1% formic acid, shaken, and centrifuged at4000 rpm and 4° C. for 10 min. 10 μL of the quenched mixture was diluted20-fold in 190 μL CH₃CN/H₂O (50/50) with 0.05% formic acid with mixing.10 μL of this 20-fold dilution mixture was then further diluted in 190μL CH₃CN/H₂O (50/50) with 0.05% formic acid for a total 800 fold dilutedmixtures. These mixtures then were analyzed for product compound (3s)formation by LC-MS in MRM mode as described in Example 3.

TABLE 3K Engineered Polypeptides and Relative Activity ImprovementsIncreased Activity¹ Relative to SEQ ID NO: 440 (1j) + (2g) → (3q) SEQ IDAssay² NO: Amino Acid Differences 44° C., pH 8.0, 15% (nt/aa) (Relativeto SEQ ID NO: 12) DMSO 605/606 A37P; A57D; V82T; T156N; G170K; E256S;V259I; Y263Q; P267S; ++ P278W; 607/608 A18G; A37P; V82T; T156N; G170K;E256S; V259I; Y263Q; + 609/610 A18G; A37P; V82T; F140M; T156N; E256S;V259I; Y263Q; A352Q; + 611/612 K26M; A37P; A57V; V82T; T156N; A158S;G170K; E256S; V259I; + Y263Q; P267N; P278S; 613/614 K26M; A37P; A57V;V82T; T156N; A158T; G170K; E256S; V259I; + Y263Q; P267S; P278W; 615/616A37P; V82T; T156N; A158R; V259I; Y263Q; P267N; + 617/618 A37P; A57D;V82T; T156N; G170S; V259I; Y263Q; P267S; P278W; + 619/620 A37P; A57D;V82T; T156N; A158T; G170S; V259I; Y263Q; P267S; + P278S; 621/622 A37P;A57D; V82T; T156N; G170K; V259I; Y263Q; P267S; P278W; + 623/624 A37P;A57V; V82T; T156N; A158V; V259I; Y263Q; P267S; + 625/626 A37P; A57D;V82T; T156N; V259I; Y263Q; P267S; P278W; + 627/628 K26M; A37P; A57V;V82T; T156N; V259I; Y263Q; P267N; P278S; + 629/630 A37P; V82T; T156N;V259I; Y263Q; P267S; P278W; + 631/632 A18G; A37P; V82T; F140M; T156N;G170K; E256S; V259I; Y263Q; + P278W; A352Q; 633/634 A18G; A37P; V82T;T156N; G170K; V259I; Y263Q; A352Q; + 635/636 A37P; V82T; T156N; G170S;V259I; Y263Q; P267S; P278W; + 637/638 K26M; A37P; V82T; T156N; G170K;V259I; Y263Q; P267S; P278W; + 639/640 A37P; A57L; V82T; T156N; A158T;V259I; Y263Q; P267S; + 641/642 A37P; A57V; V82T; T156N; G170K; V259I;Y263Q; P267S; P278W; + 643/644 K26M; A37P; A57V; V82T; T156N; G170S;V259I; Y263Q; P267N; + P278S; 645/646 K26M; A37P; A57V; V82T; T156N;V259I; Y263Q; P267S; P278S; + 647/648 A18G; A37P; V82T; T156N; V259I;Y263Q; + 649/650 A37P; V82T; T156N; E256S; V259I; Y263Q; P267N; +651/652 K26M; A37P; V82T; T156N; G170K; V259I; Y263Q; P267S; + 653/654A37P; A57V; V82T; T156N; G170S; V259I; Y263Q; P267S; P278S; + 655/656A37P; A57V; V82T; T156N; V259I; Y263Q; P267G; P278W; + 657/658 K26M;A37P; A57D; V82T; T156N; G170K; V259I; Y263Q; P267S; + 659/660 A37P;A57V; V82T; T156N; G170K; E256S; V259I; Y263Q; P278W; + 661/662 K26M;A37P; A57V; V82T; T156N; A158S; G170K; V259I; Y263Q; + P267S; P278S;663/664 A37P; A57L; V82T; T156N; G170K; V259I; Y263Q; P267S; P278W; +665/666 A37P; V82T; T156N; G170K; V259I; Y263Q; P267S; + 667/668 A18G;A37P; V82T; G126S; F140M; T156N; G170K; A234V; V259I; + Y263Q; P278W;669/670 A37P; A57V; V82T; T156N; V259I; Y263Q; P267S; + 671/672 A37P;V82T; T156N; G170K; V259I; Y263Q; P267S; P278S; + 673/674 K26M; A37P;V82T; T156N; G170S; V259I; Y263Q; P267S; P278W; + 675/676 A37P; A57D;V82T; T156N; G170K; V259I; Y263Q; P267G; P278W; + 677/678 A37P; V82T;T156N; V259I; Y263Q; P267S; P278S; + 679/680 A37P; A57D; V82T; T156N;G170K; E256I; V259I; Y263Q; P267S; + P278W; 681/682 A37P; A57V; V82T;T156N; G170K; V259I; Y263Q; P267S; + 683/684 A18G; A37P; V82T; T156N;G170K; V259I; Y263Q; P278W; A352Q; + 685/686 A37P; A57L; V82T; T156N;G170K; V259I; Y263Q; P267S; P278S; + 687/688 K26M; A37P; A57V; V82T;T156N; G170K; V259I; Y263Q; P267S; + 689/690 A37P; A57V; V82T; T156N;V259I; Y263Q; P267N; + 691/692 A37P; V82T; T156N; A158T; G170K; V259I;Y263Q; P267N; + 693/694 K26M; A37P; A57V; V82T; T156N; G170S; V259I;Y263Q; P267S; P278S; + 695/696 A37P; V82T; T156N; V259I; Y263Q; P267S; +697/698 A37P; V82T; T156N; G170S; V259I; Y263Q; P267S; P278S; + 699/700A37P; V82T; T156N; G170K; V259I; Y263Q; P267N; + 701/702 A37P; A57V;V82T; T156N; G170K; V259I; Y263Q; P267S; P278S; + 703/704 A37P; A57L;V82T; T156N; G170S; E256S; V259I; Y263Q; P267G; P278S; + 705/706 A37P;A57V; V82T; T156N; G170K; V259I; Y263Q; P267N; P278S; + 707/708 K26M;A37P; V82T; T156N; G170S; V259I; Y263Q; P267S; P278S; + ¹Levels ofincreased activity were determined relative to the reference polypeptideof SEQ ID NO: 440 and defined as follows: “+” = at least 1.2-foldincreased activity, but less than 1.8-fold increased activity; “++” = atleast 1.8-fold increased activity, but less than 2.5-fold increasedactivity; “+++” = at least 2.5-fold increased activity but less than7-fold increased activity; “++++” at least 7-fold increased activity.²Substrate Compounds (1j) + (2) → Product Compound (3q) Activity Assay(pH 8.0, 15% DMSO): Enzyme Lysate Preparation: E. coli cells expressingthe polypeptide variant gene of interest were pelleted, placed in96-well plates and lysed in 400 μL lysis buffer (1 g/L lysozyme and 0.5g/L PMBS in 0.1M phosphate buffer, pH 8.0) with low-speed shaking for 2h on titre-plate shaker at room temperature. The lysate containingplates were centrifuged at 4000 rpm and 4° C. for 20 min and the clearlysate supernatant used for assay reactions. HTP Assay Reaction: Theenzyme assay reaction was carried out in a total volume of 100 μL in a96-well plate format. The assay reaction was initiated by adding thefollowing to each well containing 55 μL of the lysate: (i) 10 μL of GDHcofactor recycling pre-mix (pre-mix contains 100 g/L glucose, 30 g/LNAD+, 10 g/L GDH-105); (ii) 20 μL of (2g) stock solution (0.5 mM); and15 μL ketone substrate (1j) stock solution (333 mM compound (1j) inDMSO). The resulting assay reaction included 50 mM ketone substratecompound (1j), 100 mM amine substrate (compound (2g)), 55.5 mM glucose,3 g/L NAD+, 1 g/L GDH-105, 100 mM potassium phosphate, pH 8.0, 15% (v/v)DMSO. The reaction plate was heat-sealed and shaken at 4000 rpmovernight (16-24 h) at 44° C. HPLC Work-up and Analysis: Each reactionmixture was quenched by adding 100 μL CH₃CN with 0.1% formic acid,shaken, and centrifuged at 4000 rpm and 4° C. for 10 min. 20 μL of thequenched mixture was diluted 5-fold in 80 μL CH₃CN/H₂O (50/50) with0.05% formic acid with mixing. 10 μL of these mixtures then wereanalyzed for product compound (3q) formation by HPLC as described inExample 4.

TABLE 3L Engineered Polypeptides and Relative Activity ImprovementsIncreased Activity¹ Relative to SEQ ID NO: 604 SEQ ID (1e) + (2d) →(3s)² NO: Amino Acid Differences 44° C., pH 8.0, 30% (nt/aa) (Relativeto SEQ ID NO: 12) DMSO 709/710 L12M; L65V; V82T; A234C; E256A; I261H;S262V; Y263C; N277L; ++ V292E; 711/712 L12M; L65V; M159Q; A234C; I242C;K260N; I261Q; S262V; Y263C; ++ N277L; S284H; V292E; 713/714 L12M; L65V;M159Q; E256A; I261Q; S262V; Y263C; N277L; V292E; ++ 715/716 L12M; L65V;M159Q; A234C; I242C; E256V; K260N; I261H; S262V; ++ Y263C; N277L; V292E;717/718 L12M; L65V; M159Q; E256A; I261H; S262V; Y263C; N277L; V292E; ++719/720 L12M; L65V; M159Q; E256V; K260N; I261H; S262V; Y263C; N277L; ++S284H; V292E; 721/722 L12M; L65V; V82T; Y153F; M159Q; I242C; I261H;S262V; Y263C; ++ N277L; S284H; V292E; 723/724 L12M; L65V; L201I; A234C;E256V; I261Q; S262V; Y263C; N277L; ++ V292E; 725/726 L12M; L65V; M159Q;A234C; E256V; K260N; I261H; S262V; Y263C; ++ N277L; S284H; V292E;727/728 L12M; L65V; K260N; I261Q; S262V; Y263C; N277L; S284H; V292E; ++729/730 L12M; L65V; K260N; I261H; S262V; Y263C; N277L; V292E; ++ 731/732L12M; L65V; M159Q; A234C; K260N; I261Q; S262V; Y263C; N277L; ++ S284H;V292E; 733/734 L12M; L65V; V82T; Y153F; A234C; I242C; E256V; I261Q;S262V; ++ Y263C; N277L; S284H; V292E; 735/736 L12M; L65V; Y153F; M159Q;L201I; A234C; E256A; I261Q; S262V; ++ Y263C; N277L; S284H; V292E;737/738 L12M; L65V; M159Q; L201I; I242C; I261H; S262V; Y263C; N277L; ++V292E; 739/740 L12M; L65V; L201I; A234C; I261Q; S262V; Y263C; N277L;S284H; ++ V292E; 741/742 L12M; L65V; M159Q; L201I; A234C; I242C; E256A;I261Q; S262V; ++ Y263C; N277L; S284H; V292E; 743/744 L12M; L65V; Y153E;M159Q; A234C; I242C; E256A; I261H; S262V; ++ Y263C; N277L; S284H; V292E;745/746 L12M; L65V; L201I; A234C; I261H; S262V; Y263C; N277L; V292E; ++747/748 L12M; L65V; Y153E; L201I; A234C; I242C; K260N; I261Q; S262V; ++Y263C; N277L; S284H; V292E; 749/750 L12M; L65V; M159Q; E256V; K260N;I261H; S262V; Y263C; N277L; ++ V292E; 751/752 L12M; L65V; M159Q; A234C;E256A; K260N; I261H; S262V; Y263C; ++ N277L; S284H; V292E; 753/754 L12M;Y153F; L201I; A234C; E256A; K260N; I261Q; S262V; Y263C; ++ N277L; S284H;V292E; 755/756 L12M; L65V; V82T; L201I; A234C; I242C; K260N; I261Q;S262V; ++ Y263C; N277L; S284H; V292E; 757/758 L12M; L65V; M159Q; I261Q;S262V; Y263C; N277L; V292E; ++ 759/760 L12M; L65V; V82T; M159Q; I242C;E256V; K260N; I261Q; S262V; ++ Y263C; N277L; V292E; 761/762 L12M; L65V;V82T; M159Q; I242C; E256A; K260N; I261Q; S262V; ++ Y263C; N277L; S284H;V292E; 763/764 L12M; L65V; V82T; Y153F; A234C; E256A; I261H; S262V;Y263C; ++ N277L; S284H; V292E; 765/766 L12M; L65V; M159Q; L201I; I261H;S262V; Y263C; N277L; V292E; ++ 767/768 L12M; L65V; M159Q; I242C; K260N;I261H; S262V; Y263C; N277L; ++ S284H; V292E; 769/770 L12M; L65V; Y153F;L201I; A234C; I242C; K260N; I261H; S262V; ++ Y263C; N277L; S284H; V292E;771/772 L12M; L65V; V82T; Y153F; A234C; E256A; I261H; S262V; Y263C; ++N277L; V292E; 773/774 L12M; L65V; V82T; M159Q; A234C; I261H; S262V;Y263C; N277L; ++ V292E; 775/776 L12M; L65V; V82T; Y153F; A234C; I242C;E256V; K260N; I261Q; ++ S262V; Y263C; N277L; S284H; V292E; 777/778 L12M;L65V; V82T; M159Q; I242C; I261H; S262V; Y263C; N277L; ++ S284H; V292E;779/780 L12M; L65V; V82T; Y153E; L201I; A234C; I242C; I261H; S262V; ++Y263C; N277L; S284H; V292E; 781/782 L12M; L65V; M159Q; L201I; I242C;E256A; I261H; S262V; Y263C; ++ N277L; V292E; 783/784 L12M; L65V; M159Q;A234C; I261Q; S262V; Y263C; N277L; S284H; ++ V292E; 785/786 L12M; L65V;L201I; K260N; I261Q; S262V; Y263C; N277L; V292E; ++ 787/788 L12M; L65V;M159Q; A234C; E256A; K260N; I261Q; S262V; Y263C; ++ N277L; S284H; V292E;789/790 L12M; M159Q; A234C; K260N; I261Q; S262V; Y263C; N277L; V292E; ++791/792 L12M; L65V; V82T; M159Q; A234C; E256V; I261Q; S262V; Y263C; +++N277L; S284H; V292E; 793/794 L12M; L65V; M159Q; L201I; I261Q; S262V;Y263C; N277L; V292E; ++ 795/796 L12M; L65V; V82T; M159Q; L201I; A234C;I261H; S262V; Y263C; ++ N277L; V283M; S284H; V292E; 797/798 L12M; L65V;Y153F; A234C; K260N; I261Q; S262V; Y263C; N277L; ++ S284H; V292E;799/800 L12M; L65V; M159Q; L201I; A234C; E256A; I261Q; S262V; Y263C; ++N277L; S284H; V292E; 801/802 L12M; L65V; M159Q; A234C; I242C; E256A;K260N; I261Q; S262V; ++ Y263C; N277L; S284H; V292E; 803/804 L12M; L65V;Y153F; L201I; A234C; K260N; I261Q; S262V; Y263C; ++ N277L; S284H; V292E;805/806 L12M; L65V; M159Q; A234C; E256A; K260N; I261Q; S262V; Y263C; +++N277L; V292E; 807/808 L12M; L65V; V82T; Y153E; L201I; A234C; I242C;E256V; K260N; +++ I261H; S262V; Y263C; N277L; S284H; V292E; 809/810L12M; L65V; Y153E; L201I; A234C; E256A; I261Q; S262V; Y263C; +++ N277L;V292E; 811/812 L12M; L65V; M159Q; K260N; I261H; S262V; Y263C; N277L;V292E; +++ 813/814 L12M; L65V; V82T; M159Q; A234C; I242C; E256V; K260N;I261H; +++ S262V; Y263C; N277L; S284H; V292E; 815/816 L12M; L65V; V82T;M159Q; A234C; E256V; K260N; I261H; S262V; +++ Y263C; N277L; S284H;V292E; 817/818 L12M; L65V; M159Q; L201I; I242C; E256A; I261Q; S262V;Y263C; +++ N277L; S284H; V292E; 819/820 L12M; L65V; V82T; M159Q; A234C;I242C; K260N; I261H; S262V; +++ Y263C; N277L; V292E; 821/822 L12M; L65V;V82T; M159Q; L201I; I242C; E256V; I261Q; S262V; +++ Y263C; N277L; S284H;V292E; 823/824 L12M; L65V; Y153E; L201I; A234C; I261Q; S262V; Y263C;N277L; +++ V292E; 825/826 L12M; L65V; M159Q; A234C; K260N; I261H; S262V;Y263C; N277L; +++ S284H; V292E; 827/828 L12M; L65V; V82T; Y153E; L201I;A234C; I242C; E256A; I261Q; +++ S262V; Y263C; N277L; V292E; 829/830L12M; L65V; L201I; A234C; E256A; I261Q; S262V; Y263C; N277L; +++ S284H;V292E; 831/832 L12M; L65V; Y153E; M159Q; A234C; I242C; K260N; I261H;S262V; +++ Y263C; N277L; S284H; V292E; 833/834 L12M; L65V; Y153E; M159Q;A234C; I242C; E256A; K260N; I261Q; +++ S262V; Y263C; N277L; V292E;835/836 L12M; L65V; V82T; Y153F; L201I; A234C; I242C; E256A; I261Q; +++S262V; Y263C; N277L; S284H; V292E; 837/838 L12M; L65V; V82T; Y153E;L201I; A234C; I242C; E256A; K260N; +++ I261Q; S262V; Y263C; N277L;S284H; V292E; 839/840 L12M; L65V; V82T; Y153F; L201I; A234C; E256A;I261Q; S262V; +++ Y263C; N277L; S284H; V292E; 841/842 L12M; L65V; V82T;Y153F; A234C; E256V; K260N; I261Q; S262V; +++ Y263C; N277L; S284H;V292E; 843/844 L12M; L65V; V82T; M159Q; A234C; I261Q; S262V; Y263C;N277L; +++ S284H; V292E; 845/846 L12M; L65V; M159Q; L201I; A234C; I242C;K260N; I261H; S262V; +++ Y263C; N277L; S284H; V292E; 847/848 L12M; L65V;V82T; L201I; A234C; I242C; E256V; I261Q; S262V; +++ Y263C; N277L; S284H;V292E; 849/850 L12M; L65V; V82T; M159Q; A234C; K260N; I261Q; S262V;Y263C; +++ N277L; S284H; V292E; 851/852 L12M; L65V; V82T; M159Q; A234C;E256V; I261H; S262V; Y263C; +++ N277L; S284H; V292E; 853/854 L12M; L65V;V82T; M159Q; A234C; I242C; E256A; K260N; I261Q; +++ S262V; Y263C; N277L;S284H; V292E; 855/856 L12M; L65V; M159Q; L201I; A234C; I261H; S262V;Y263C; N277L; +++ V292E; 857/858 L12M; L65V; V82T; L201I; A234C; E256A;K260N; I261H; S262V; +++ Y263C; N277L; V292E; 859/860 L12M; L65V; V82T;Y153F; M159Q; A234C; I261Q; S262V; Y263C; +++ N277L; S284H; V292E;861/862 L12M; L65V; V82T; A234C; I242C; E256V; K260N; I261H; S262V; +++Y263C; N277L; V292E; 863/864 L12M; L65V; L201I; A234C; I242C; K260N;I261Q; S262V; Y263C; +++ N277L; S284H; V292E; 865/866 L12M; L65V; Y153E;M159Q; A234C; I242C; K260N; I261Q; S262V; +++ Y263C; N277L; S284H;V292E; 867/868 L12M; L65V; M159Q; L201I; I242C; K260N; I261Q; S262V;Y263C; +++ N277L; S284H; V292E; 869/870 L12M; L65V; M159Q; L201I; A234C;E256A; I261H; S262V; Y263C; +++ N277L; S284H; V292E; 871/872 L12M; L65V;M159Q; L201I; A234C; I242C; E256A; K260N; I261Q; +++ S262V; Y263C;N277L; V292E; 873/874 L12M; L65V; M159Q; L201I; A234C; I261H; S262V;Y263C; N277L; +++ S284H; V292E; 875/876 L12M; L65V; V82T; L201I; I242C;E256V; K260N; I261Q; S262V; +++ Y263C; N277L; S284H; V292E; 877/878L12M; L65V; M159Q; A234C; K260N; I261H; S262V; Y263C; N277L; +++ V292E;879/880 L12M; L65V; V82T; M159Q; L201I; A232G; A234C; I242C; E256V; +++K260N; I261H; S262V; Y263C; N277L; S284H; V292E; 881/882 L12M; L65V;V82T; M159Q; L201I; I242C; K260N; I261Q; S262V; +++ Y263C; N277L; S284H;V292E; 883/884 L12M; L65V; M159Q; L201I; I242C; E256A; K260N; I261Q;S262V; +++ Y263C; N277L; S284H; V292E; 885/886 L12M; L65V; V82T; M159Q;L201I; A234C; I242C; E256A; I261H; +++ S262V; Y263C; N277L; S284H;V292E; 887/888 L12M; L65V; V82T; M159Q; A234C; I242C; E256V; K260N;I261Q; +++ S262V; Y263C; N277L; S284H; V292E; 889/890 L12M; L65V; V82T;Y153F; L201I; A234C; I242C; E256A; I261H; +++ S262V; Y263C; N277L;S284H; V292E; 891/892 L12M; L65V; V82T; Y153F; L201I; I242C; K260N;I261Q; S262V; +++ Y263C; N277L; S284H; V292E; 893/894 L12M; V82T; M159Q;L201I; A234C; I242C; E256A; I261H; S262V; +++ Y263C; N277L; S284H;V292E; 895/896 L12M; L65V; M159Q; L201I; A234C; I242C; K260N; I261Q;S262V; +++ Y263C; N277L; S284H; V292E; 897/898 L12M; L65V; M159Q; L201I;A234C; I261Q; S262V; Y263C; N277L; +++ S284H; V292E; 899/900 L12M; L65V;Y153F; M159Q; L201I; A234C; E256A; K260N; I261H; +++ S262V; Y263C;N277L; S284H; V292E; 901/902 L12M; L65V; V82T; Y153F; L201I; A234C;E256V; K260N; I261Q; +++ S262V; Y263C; N277L; S284H; V292E; 903/904L12M; V82T; M159Q; L201I; A234C; I242C; E256A; I261Q; S262V; +++ Y263C;N277L; S284H; V292E; 905/906 L12M; L65V; V82T; Y153F; L201I; A234C;I242C; E256V; K260N; +++ I261Q; S262V; Y263C; N277L; S284H; V292E;907/908 L12M; V82T; Y153E; M159Q; L201I; A234C; I242C; E256A; K260N; +++I261Q; S262V; Y263C; N277L; S284H; V292E; 909/910 L12M; L65V; V82T;Y153F; M159Q; L201I; A234C; I242C; E256V; +++ K260N; I261Q; S262V;Y263C; N277L; S284H; V292E; 911/912 L12M; L65V; V82T; M159Q; L201I;A234C; I242C; E256V; K260N; +++ I261H; S262V; Y263C; N277L; S284H;V292E; 913/914 L12M; L65V; V82T; Y153F; L201I; A234C; I261H; S262V;Y263C; +++ N277L; V292E; 915/916 L12M; L65V; M159Q; L201I; A234C; K260N;I261H; S262V; Y263C; +++ N277L; S284H; V292E; 917/918 L12M; L65V; V82T;M159Q; L201I; A234C; E256V; K260N; I261H; +++ S262V; Y263C; N277L;S284H; V292E; 919/920 L12M; L65V; V82T; M159Q; L201I; A234C; E256A;K260N; I261H; +++ S262V; Y263C; N277L; S284H; V292E; 921/922 L12M; L65V;V82T; M159Q; L201I; I242C; E256A; K260N; I261Q; +++ S262V; Y263C; N277L;V292E; 923/924 L12M; L65V; V82T; M159Q; L201I; A234C; E256A; K260N;I261H; +++ S262V; Y263C; N277L; V292E; ¹Levels of increased activitywere determined relative to the reference polypeptide of SEQ ID NO: 604and defined as follows: “+” = at least 1.2-fold increased activity, butless than 1.8-fold increased activity; “++” = at least 1.8-foldincreased activity, but less than 2.5-fold increased activity; “+++” =at least 2.5-fold increased activity but less than 7-fold increasedactivity; “++++” at least 7-fold increased activity. ²SubstrateCompounds (1e) + (2d) → Product Compound (3s) Activity Assay: EnzymeLysate Preparation: E. coli cells expressing the polypeptide variantgene of interest were pelleted, placed in 96-well plates and lysed in250 μL lysis buffer (1 g/L lysozyme and 0.5 g/L PMBS in 0.1M phosphatebuffer, pH 8.0) with low-speed shaking for 2 h on titre-plate shaker atroom temperature. The lysate containing plates were centrifuged at 4000rpm and 4° C. for 20 min and the clear lysate supernatant used for assayreactions. HTP Assay Reaction: The enzyme assay reaction was carried outin a total volume of 100 μL in a 96-well plate format. The assayreaction was initiated by adding the following to each well containing50 μL of the lysate: (i) 20 μL of GDH cofactor recycling pre-mix(pre-mix contains 50 g/L glucose, 15 g/L NAD+, 5 g/L GDH-105); (ii) 15μL of aniline stock solution (667 mM in DMSO); and 15 μL ketonesubstrate stock solution (333 mM compound (1e) in DMSO). The resultingassay reaction included 50 mM ketone substrate compound (1e), 100 mMamine substrate (compound (2d)), 55.5 mM glucose, 3 g/L NAD+, 1 g/LGDH-105, 100 mM potassium phosphate, pH 8.0, 15% (v/v) or 30% (v/v)DMSO. The reaction plate was heat-sealed and shaken at 4000 rpmovernight (16-24 h) at 44° C. Work-up and Analysis: Each reactionmixture was quenched by adding 100 μL CH₃CN with 0.1% formic acid,shaken, and centrifuged at 4000 rpm and 4° C for 10 min. 10 μL of thequenched mixture was diluted 20-fold in 190 μL CH₃CN/H₂O (50/50) with0.05% formic acid with mixing. 10 μL of this 20-fold dilution mixturewas then further diluted in 190 μL CH₃CN/H₂O (50/50) with 0.05% formicacid for a total 800 fold diluted mixtures. These mixtures then wereanalyzed for product compound (3s) formation by LC-MS in MRM mode asdescribed in Example 4.

From an analysis of the exemplary polypeptides shown in Tables 3A-3L,improvements in enzyme properties are associated with residuedifferences as compared to the reference sequence of the engineeredpolypeptide of SEQ ID NO:6 at residue positions X12, X18, X20, X26, X27,X29, X37, X57, X65, X74, X82, X87, X93, X94, X96, X108, X111, X126,X138, X140, X141, X142, X143, X153, X154, X156, X157, X158, X159, X163,X170, X175, X177, X195, X197, X200, X201, X220, X221, X223, X234, X241,X242, X253, X254, X256, X257, X259, X260, X261, X262, X263, X264, X265,X267, X270, X272, X273, X274, X276, X277, X278, X279, X281, X282, X283,X284, X291, X292, X295, X296, X326, and X352. The specific residuedifferences at each of these positions that are associated with theimproved properties include: X12M, X18G, X20V, X26M/V, X27S, X29K, X37P,X57D/L/V, X65I/V, X74W, X82C/P/T, X87A, X93G/Y, X94N, X96C, X108S,X111A/H, X126S, X138L, X140M, X141M/N/W, X142A, X143F/L/W/Y, X153E/F/Y,X154C/D/F/G/K/L/N/Q/S/TN/Y, X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V,X159C/L/Q/V, X163V, X170F/K/R/S, X175R, X177R, X195S, X197V, X200S,X201I, X220C/K/Q, X221F, X223S, X234V/C/L, X241K, X242C/L, X253K/N,X254R, X256A/E/I/L/S/T/V, X257Q, X259C/I/L/M/R/T, X260A/D/G/N/Q/V/Y,X261E/F/H/L/P/Q/R/Y, X262F/G/P/V, X263C/D/E/H/M/L/M/N/P/Q/V, X264V,X265L, X267E/G/H/I/N/S, X270L, X272D, X273C/W, X274L/M/S, X276L,X277A/H/I/L, X278E/H/K/N/R/S/W, X279L/T, X281A, X282A/R, X283M/V,X284C/F/H/L/P/Q/S, X291E, X292E/P, X295F, X296N, X326V, and X352Q.

The specific enzyme properties associated with the residue differencesas compared to SEQ ID NO: 6 at the residue positions above include,among others, enzyme activity, and stability (thermal and solvent).Substantial improvements in enzyme activity and stability are associatedwith residue differences at residue positions X12, X82, X94, X111, X141,X143, X153, X154, X159, X163, X256, X259, X273, X274, X283, X284, andX296, and with the specific residue differences X12M, X82C/P/T, X94N,X111A/H, X141M/N/W, X143F/L/W/Y, X153E/F/Y, X154C/D/G/F/K/L/N/Q/S/TN/Y,X159C/L/Q/V, X163V, X256A/E/I/L/S/T/V, X259C/I/L/M/R/T, X273C/W,X274L/M/S, X283V, X284C/F/H/L/P/Q/S, and X296N/V. In particular, theamino acid residue differences X12M, X82C/P/T, and X111A/H, provideincreased imine reductase activity and/or stability across a range ofketone and amine substrates as shown by the results in Tables 3A-3L.

Further improvements in activity, stability, and selectivity for thevarious combinations of unactivated ketone and unactivated aminesubstrate compounds in producing the various amine product compounds(3o), (3p), (3q), (3r), and (3s) (e.g., reactions (o)-(s) of Table 2)are associated with residue differences at residue positions: X18, X20,X26, X27, X29, X37, X57, X65, X74, X87, X93, X96, X108, X126, X138,X140, X142, X156, X157, X158, X170, X175, X177, X195, X197, X200, X201,X220, X221, X223, X234, X241, X242, X253, X254, X257, X260, X261, X262,X263, X264, X265, X267, X270, X272, X276, X277, X278, X279, X281, X282,X291, X292, X295, X326, and X352, and includes the specific amino acidresidue differences X18G, X20V, X26M/V, X27S, X29K, X37P, X57D/L/V,X65I/V, X74W, X87A, X93G/Y, X96C, X108S, X126S, X138L, X140M, X142A,X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V, X170F/K/R/S, X175R, X177R,X195S, X197V, X200S, X201I, X220C/K/Q, X221F, X223S, X234V/C/L, X241K,X242C/L, X253K/N, X254R, X257Q, X260A/D/G/N/Q/V/Y, X261E/F/H/L/P/Q/R/Y,X262F/G/P/V, X263C/D/E/H/M/L/M/N/P/Q/V, X264V, X265L, X267E/G/H/I/N/S,X270L, X272D, X276L, X277A/H/I/L, X278E/H/K/N/R/S/W, X279L/T, X281A,X282A/R, X291E, X292E/P, X295F, X326V, and X352Q. Accordingly, theresidue differences at the foregoing residue positions can be usedindividually or in various combinations to produce engineered iminereductase polypeptides having the desired improved properties,including, among others, enzyme activity, stability, selectivity, andsubstrate tolerance.

Additionally, as noted above, the crystal structure of the opinedehydrogenase CENDH has been determined (See e.g., Britton et al., Nat.Struct. Biol., 5:593-601 [1998]). Accordingly, this correlation of thevarious amino acid differences and functional activity disclosed hereinalong with the known three-dimensional structure of the wild-type enzymeCENDH can provide the ordinary artisan with sufficient information torationally engineer further amino acid residue changes to thepolypeptides provided herein (and to homologous opine dehydrogenaseenzymes including OpDH, BADH, CEOS, and TauDH), and retain or improve onthe imine reductase activity or stability properties. In someembodiments, it is contemplated that such improvements can includeengineering the engineered polypeptides of the present invention to haveimine reductase activity with a range of substrates and provide a rangeof products as described in Scheme 1.

In light of the guidance provided herein, it is further contemplatedthat any of the exemplary engineered polypeptide sequences ofeven-numbered sequence identifiers SEQ ID NOS:8-924, can be used as thestarting amino acid sequence for synthesizing other engineered iminereductase polypeptides, for example by subsequent rounds of evolution byadding new combinations of various amino acid differences from otherpolypeptides in Tables 3A-3L, and other residue positions describedherein. Further improvements may be generated by including amino aciddifferences at residue positions that had been maintained as unchangedthroughout earlier rounds of evolution. Accordingly, in someembodiments, the engineered polypeptide having imine reductase activitycomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the reference sequence of SEQ ID NO:6 and at leastone of the following features:

(i) a residue difference as compared to the reference sequence of SEQ IDNO:6 at a position selected from X12, X18, X26, X27, X57, X65, X87, X93,X96, X126, X138, X140, X142, X159, X170, X175, X177, X195, X200, X221,X234, X241, X242, X253, X254, X257, X262, X263, X267, X272, X276, X277,X278, X281, X282, X291, and X352, optionally wherein the residuedifference at the position is selected from X12M, X18G, X26M/V, X27S,X57D/L/V, X65I/V, X87A, X93G/Y, X96C, X126S, X138L, X140M, X142A,X159C/L/Q/V, X170F/K/R/S, X175R, X177R, X195S, X200S, X221F, 234C/L,X241K, X242C/L, X253K/N, X254R, X257Q, X262F/G/P/V,X263C/D/E/H/M/L/M/N/P/Q/V, X267E/G/H/I/N/S, X272D, X276L, X277H/L,X278E/H/K/N/R/S/W, X281A, X282A/R, X291E, and X352Q;

(ii) a residue difference as compared to the reference sequence of SEQID NO:6 selected from X20V, X29K, X37P, X74W, X82C/T, X94N, X108S,X111A/H, X141M/N, X143F/L/Y, X153E/F, X154C/D/G/K/L/N/S/T/V,X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V, X163V, X197V, X201I,X220C/K/Q, X223S, X256A/E/I/L/S/T, X259C/R, X260A/D/N/Q/V/Y,X261E/F/H/L/P/Q/Y, X264V, X270L, X273C, X274L/S, X279T, X284C/F/H/P/Q/S,X292E/P, and X295F; and/or

(iii) two or more residue differences as compared to the referencesequence of SEQ ID NO:6 selected from X82P, X141W, X153Y, X154F,X259I/L/M, X274L/M, X283V, and X296N/V.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence comprising at least oneresidue difference as compared to the reference sequence of SEQ ID NO:6selected from X12M, X37P, X82T, X111A, X154S, X156N/M, X223S, X256E,X260D, X261H, X262P, X263C/E/Q, X267G, X277L, X281A, X284P/S, and X292E.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence comprising at least oneresidue difference as compared to the reference sequence of SEQ ID NO:6selected from X93G/Y, X94N, X96C, X111A/H, X142A, X159L, X163V, X256E,X259R, X273C, and X284P/S.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence comprising at least tworesidue differences as compared to the reference sequence of SEQ ID NO:6selected from X82P, X141W, X143W, X153Y, X154F/Q/Y, X256V, X259I/L/M/T,X260G, X261R, X265L, X273W, X274M, X277A/I, X279L, X283V, X284L, X296N,X326V. In some embodiments, the at least two residue differences areselected from X141W, X153Y, X154F, X259I/L/M, X274L/M, X283V, andX296N/V.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence comprising at least acombination of residue differences as compared to the reference sequenceof SEQ ID NO:6 selected from: (a) X153Y, and X283V; (b) X141W, X153Y,and X283V; (c) X141W, X153Y, X274L/M, and X283V; (d) X141W, X153Y,X154F, X274L/M, and X283V; (e) X141W, X153Y, X154F, and X283V; (f)X141W, X153Y, X283V, and X296N/V; (g) X141W, X153Y, X274L/M, X283V, andX296N/V; (h) X111A, X153Y, X256E, X274M, and X283V; (i) X111A, X141W,X153Y, X273C, X274M, X283V, and X284S; (j) X111A, X141W, X153Y, X273C,and X283V; (k) X111A, X141W, X153Y, X154F, X256E, X274M, X283V, X284S,and X296N; (l) X111A, X141W, X153Y, X256E, X273W, X274L, X283V, X284S,and X296N; (m) X111H, X141W, X153Y, X273W, X274M, X284S, and X296N; (n)X111H, X141W, X153Y, X154F, X273W, X274L, X283V, X284S, and X296N; (o)X82P, X141W, X153Y, X256E, X274M, and X283V; (p) X82P, X111A, X141W,X153Y, X256E, X274M, X283V, M284S, and E296V; (q) X94N, X143W, X159L,X163V, X259M, and X279L; (r) X141W, X153Y, X154F, and X256E; and (s)X153Y, X256E, and X274M.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence comprising at least one of theabove combinations of amino acid residue differences (a)-(s), andfurther comprises at least one residue difference as compared to thereference sequence of SEQ ID NO:6 selected from X12M, X18G, X20V,X26M/V, X27S, X29K, X37P, X57D/L/V, X65I/V, X74W, X82C/T, X87A, X93G/Y,X94N, X96C, X108S, X111A/H, X126S, X138L, X140M, X141M/N, X142A,X143F/L/Y, X153E/F, X154C/D/G/K/L/N/S/T/V, X156H/L/N/M/R, X157F/Q/T/Y,X158I/L/R/S/T/V, X159C/L/Q/V, X163V, X170F/K/R/S, X175R, X177R, X195S,X197V, X200S, X201I, X220C/K/Q, X221F, X223S, X234V/C/L, X241K, X242C/L,X253K/N, X254R, X256A/E/I/L/S/T, X257Q, X259C/R, X260A/D/N/Q/V/Y,X261E/F/H/L/P/Q/Y, X262P, X262F/G/V, X263C/D/E/H/M/L/M/N/P/Q/V, X264V,X267E/G/H/I/N/S, X270L, X272D, X273C, X274L/S, X276L, X277H/L,X278E/H/K/N/R/S/W, X279T, X281A, X282A/R, X284C/F/H/P/Q/S, X291E,X292E/P, X295F, and X352Q.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises the amino acid sequence comprises the combination ofresidue differences X111A, X141W, X153Y, X154F, X256E, X274M, X283V,X284S, and X296N and at least a residue difference or a combination ofresidue differences as compared to the reference sequence of SEQ ID NO:6selected from: (a) X156N; (b) X37P, X82T, and X156N; (c) X37P, X82T,X156N, and X259I; (d) X259L/M; (e) X82T, X156N, X223S, X259L, X267G, andX281A; (f) X263C; (g) X12M, X261H, X263C, X277L, and X292E; (h) X154S;and (i) X154S, X156M, X260D, X261H, X262P, X263E, and X284P.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence having 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or greater identity to a sequence ofeven-numbered sequence identifiers SEQ ID NOS:8-924.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence having 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or greater identity to a sequence ofeven-numbered sequence identifiers SEQ ID NOS:6-924, wherein the aminoacid sequence comprises an amino acid residue difference as disclosedabove (and elsewhere herein) but which does not include a residuedifference as compared to the reference sequence of SEQ ID NO:6 at oneor more residue positions selected from X29, X137, X157, X184, X197,X198, X201, X220, X232, X261, X266, X279, X280, X287, X288, X293, X295,X311, X324, X328, X332, and X353.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence having 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or greater identity to a sequence ofeven-numbered sequence identifiers SEQ ID NOS:6-924, wherein the aminoacid sequence comprises an amino acid residue difference as disclosedabove (and elsewhere herein), wherein the amino acid sequence furthercomprises a residue difference as compared to the reference sequence ofSEQ ID NO: 6 selected from: X4H/L/R, X5T, X14P, X20T, X29R/T, X37H,X67A/D, X71C/V, X74R, X82P, X94K/R/T, X97P, X100W, X111M/Q/R/S, X124L/N,X136G, X137N, X141W, X143W, X149L, X153E/V/Y, X154F/M/Q/Y,X156G/I/Q/S/T/V, X157D/H/L/M/N/R, X158K, X160N, X163T, X177C/H, X178E,X183C, X184K/Q/R, X185V, X186K/R, X197I/P, X198A/E/H/P/S, X201L,X220D/H, X223T, X226L, X232G/A/R, X243G, X246W, X256V, X258D,X259E/H/I/L/M/S/T/V/W, X260G, X261A/G/M/R/S/T, X265G/L/Y, X266T, X270G,X273W, X274M, X277A/I, X279F/L/V/Y, X280L, X283M/V, X284K/L/M/Y,X287S/T, X288G/S, X292C/G/I/P/S/TN/Y, X293H/M/L/N/Q/TN, X294A/I/V,X295R/S, X296L/N/V/W, X297A, X308F, X311C/T/V, X323C/I/M/TN, X324L/T,X326V, X328A/G/E, X332V, X353E, and X356R.

In some embodiments, the engineered polypeptide having imine reductaseactivity with improved properties as compared to SEQ ID NO:6, comprisesan amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to areference sequence selected from SEQ ID NO:6, 12, 84, 92, 146, 162, 198,228, 250, 354, and 440, and one or more residue differences as comparedto SEQ ID NO:6 at residue positions selected from: X12, X18, X20, X26,X27, X29, X37, X57, X65, X74, X82, X87, X93, X94, X96, X108, X111, X126,X138, X140, X141, X142, X143, X153, X154, X156, X157, X158, X159, X163,X170, X175, X177, X195, X197, X200, X201, X220, X221, X223, X234, X241,X242, X253, X254, X256, X257, X259, X260, X261, X262, X263, X264, X265,X267, X270, X272, X273, X274, X276, X277, X278, X279, X281, X282, X283,X284, X291, X292, X295, X296, X326, and X352.

As will be appreciated by the skilled artisan, in some embodiments, oneor a combination of residue differences above that is selected can bekept constant in the engineered imine reductases as a core sequence, andadditional residue differences at other residue positions incorporatedinto the core sequence to generate additional engineered imine reductasepolypeptides with improved properties. Accordingly, it is to beunderstood for any engineered imine reductase containing one or a subsetof the residue differences above, the present invention contemplatesother engineered imine reductases that comprise the one or subset of theresidue differences, and additionally one or more residue differences atthe other residue positions disclosed herein. By way of example and notlimitation, an engineered imine reductase comprising a residuedifference at residue position X256, can further incorporate one or moreresidue differences at the other residue positions (e.g., X111, X141,X153, X154, X198, X274, X283, X284, and X296). Indeed, the engineeredimine reductase polypeptide of SEQ ID NO:12 which comprises thecombination of residue differences as compared to SEQ ID NO:6: X111A,X141W, X153Y, X154F, X256E, X274M, X283V, X284S, and X296N, was furtherevolved to generate additional engineered imine reductase polypeptideswith improved activity and stability. These further improved engineeredimine reductase polypeptides comprise one or more residue differences ascompared to the sequence of SEQ ID NO:6 at residue positions selectedfrom X12, X18, X20, X26, X27, X29, X37, X57, X65, X74, X82, X87, X93,X94, X96, X108, X111, X126, X138, X140, X141, X142, X143, X153, X154,X156, X157, X158, X159, X163, X170, X175, X177, X195, X197, X200, X201,X220, X221, X223, X234, X241, X242, X253, X254, X256, X257, X259, X260,X261, X262, X263, X264, X265, X267, X270, X272, X273, X274, X276, X277,X278, X279, X281, X282, X283, X284, X291, X292, X295, X296, X326, andX352. The specific amino acid residue differences at these positionsassociated with improved activity or stability are selected from X12M,X18G, X20V, X26M/V, X27S, X29K, X37P, X57D/L/V, X65I/V, X74W, X82C/P/T,X87A, X93G/Y, X94N, X96C, X108S, X111A/H, X126S, X138L, X140M,X141M/N/W, X142A, X143F/L/W/Y, X153E/F/Y, X154C/D/F/G/K/L/N/Q/S/TN/Y,X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V, X159C/L/Q/V, X163V,X170F/K/R/S, X175R, X177R, X195S, X197V, X200S, X201I, X220C/K/Q, X221F,X223S, X234V/C/L, X241K, X242C/L, X253K/N, X254R, X256A/E/I/L/S/T/V,X257Q, X259C/I/L/M/R/T, X260A/D/G/N/Q/V/Y, X261E/F/H/L/P/Q/R/Y,X262F/G/P/V, X263C/D/E/H/M/L/M/N/P/Q/V, X264V, X265L, X267E/G/H/I/N/S,X270L, X272D, X273C/W, X274L/M/S, X276L, X277A/H/I/L, X278E/H/K/N/R/S/W,X279L/T, X281A, X282A/R, X283M/V, X284C/F/H/L/P/Q/S, X291E, X292E/P,X295F, X296N, X326V, and X352Q.

Accordingly, in some embodiments the engineered polypeptide having iminereductase activity comprises an amino acid sequence having at least 80%sequence identity to SEQ ID NO:6 (or any of the exemplary engineeredpolypeptides of SEQ ID NOS:8-924), one or more residue differences ascompared to the sequence of SEQ ID NO:6 at residue positions selectedfrom X111, X141, X153, X154, X256, X274, X283, X284, and X296 (asdescribed above), and further comprises one or more residue differencesas compared to the sequence of SEQ ID NO:6 at residue positions selectedfrom X12, X18, X20, X26, X27, X29, X37, X57, X65, X74, X82, X87, X93,X94, X96, X108, X111, X126, X138, X140, X141, X142, X143, X153, X154,X156, X157, X158, X159, X163, X170, X175, X177, X195, X197, X200, X201,X220, X221, X223, X234, X241, X242, X253, X254, X256, X257, X259, X260,X261, X262, X263, X264, X265, X267, X270, X272, X273, X274, X276, X277,X278, X279, X281, X282, X283, X284, X291, X292, X295, X296, X326, andX352. In some embodiments, these further residue differences areselected from X12M, X18G, X20V, X26M/V, X27S, X29K, X37P, X57D/L/V,X65I/V, X74W, X82C/P/T, X87A, X93G/Y, X94N, X96C, X108S, X111A/H, X126S,X138L, X140M, X141M/N/W, X142A, X143F/L/W/Y, X153E/F/Y,X154C/D/F/G/K/L/N/Q/S/TN/Y, X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V,X159C/L/Q/V, X163V, X170F/K/R/S, X175R, X177R, X195S, X197V, X200S,X201I, X220C/K/Q, X221F, X223S, X234V/C/L, X241K, X242C/L, X253K/N,X254R, X256A/E/I/L/S/T/V, X257Q, X259C/I/L/M/R/T, X260A/D/G/N/Q/V/Y,X261E/F/H/L/P/Q/R/Y, X262F/G/P/V, X263C/D/E/H/M/L/M/N/P/Q/V, X264V,X265L, X267E/G/H/I/N/S, X270L, X272D, X273C/W, X274L/M/S, X276L,X277A/H/I/L, X278E/H/K/N/R/S/W, X279L/T, X281A, X282A/R, X283M/V,X284C/F/H/L/P/Q/S, X291E, X292E/P, X295F, X296N, X326V, and X352Q.

Generally, the engineered polypeptides having imine reductase activityof the present invention are capable of converting a compound of formula(I) and an compound of formula (II) to an amine product compound offormula (III) (as illustrated by Scheme 1) with improved activity and/orimproved stereoselectivity relative to the Arthrobacter sp. strain C1wild-type opine dehydrogenase reference polypeptide of SEQ ID NO:2, orrelative to a reference polypeptide having imine reductase activityselected from the engineered polypeptides of even-numbered sequenceidentifiers SEQ ID NOS:8-924. In some embodiments, the improved activityand/or improved stereoselectivity is with respect to the conversion of aspecific combination of a compound of formula (I) and a compound offormula (II) shown in Table 2 to the corresponding amine productcompound of formula (III) shown in Table 2.

Accordingly, in some embodiments, the engineered polypeptides havingimine reductase activity of the present invention which have an aminoacid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a referencesequence selected from even-numbered sequence identifiers SEQ IDNOS:8-924, and one or more residue differences as compared to SEQ IDNO:6 at residue positions selected from: X12, X18, X20, X26, X27, X29,X37, X57, X65, X74, X82, X87, X93, X94, X96, X108, X111, X126, X138,X140, X141, X142, X143, X153, X154, X156, X157, X158, X159, X163, X170,X175, X177, X195, X197, X200, X201, X220, X221, X223, X234, X241, X242,X253, X254, X256, X257, X259, X260, X261, X262, X263, X264, X265, X267,X270, X272, X273, X274, X276, X277, X278, X279, X281, X282, X283, X284,X291, X292, X295, X296, X326, and X352, are capable of one or more ofthe following conversion reactions, under suitable reaction conditions,with improved activity and/or improved stereoselectivity relative to areference polypeptide of even-numbered sequence identifiers SEQ IDNOS:4-100 and 112-750:

(a) conversion of substrate compounds (1a) and (2a) to product compound(3a);

(b) conversion of substrate compounds (1a) and (2b) to product compound(3b);

(c) conversion of substrate compounds (1b) and (2a) to product compound(3c);

(d) conversion of substrate compounds (1b) and (2b) to product compound(3d);

(e) conversion of substrate compounds (1b) and (2c) to product compound(3e);

(f) conversion of substrate compounds (1b) and (2d) to product compound(3f);

(g) conversion of substrate compounds (1c) and (2a) to product compound(3g);

(h) conversion of substrate compounds (1d) and (2a) to product compound(3h);

(i) conversion of substrate compounds (1e) and (2b) to product compound(3i);

(j) conversion of substrate compounds (1f) and (2b) to product compound(3j);

(k) conversion of substrate compounds (1g) and (2e) to product compound(3k);

(l) conversion of substrate compounds (1b) and (2f) to product compound(3l);

(m) conversion of substrate compounds (1h) and (2a) to product compound(3m);

(n) conversion of substrate compounds (1i) and (2b) to product compound(3n);

(o) conversion of substrate compounds (1j) and (2b) to product compound(3o);

(p) conversion of substrate compounds (1j) and (2c) to product compound(3p);

(q) conversion of substrate compounds (1j) and (2g) to product compound(3q);

(r) conversion of substrate compounds (1i) and (2h) to product compound(3r); and

(s) conversion of substrate compounds (1e) and (2d) to product compound(3s).

In some embodiments, the engineered polypeptide having imine reductaseactivity and capable of catalyzing one or more of the above conversionreactions (a)-(s), under suitable reaction conditions, with improvedactivity and/or stereoselectivity comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to one of even-numbered sequenceidentifiers SEQ ID NOS:6-924, and the amino acid residue differences ascompared to SEQ ID NO:6 present in any one of even-numbered sequenceidentifiers SEQ ID NOS: 8-924, as provided in Tables 3A-3L.

In some embodiments, the engineered polypeptide having imine reductaseactivity and capable of catalyzing one or more of the above conversionreactions (a)-(s), under suitable reaction conditions, with improvedactivity and/or stereoselectivity has an amino acid sequence comprisinga sequence selected from the even-numbered sequence identifiers SEQ IDNOS:8-924. The wild-type opine dehydrogenase from Arthrobacter sp.strain C1 (CENDH) of SEQ ID NO:2 from which the engineered polypeptidesof the present invention were derived has no detectable activity inconverting a ketone substrate of compound (1b) and an amine substrate ofcompound (2b) to a secondary amine product compound (3d). In someembodiments, however, the engineered polypeptides having imine reductaseactivity are capable of converting a ketone substrate of compound (1b)and an amine substrate of compound (2b) to a secondary amine productcompound (3d).

Further, in some embodiments, the engineered polypeptide having iminereductase activity disclosed herein is capable of converting the ketonesubstrate of compound (1j) and the amine substrate of compound (2b) tothe amine product compound (3o) with at least 1.2 fold, 1.5 fold, 2fold, 3 fold, 4 fold, 5 fold, 10 fold or more activity relative to theactivity of the reference polypeptide of SEQ ID NO:6, or 12. In someembodiments, the engineered polypeptide having imine reductase activitydisclosed herein is capable of converting the ketone substrate ofcompound (1j) and the amine substrate of compound (2c) to the amineproduct compound (3p) with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold,4 fold, 5 fold, 10 fold or more activity relative to the activity of thereference polypeptide of SEQ ID NO:6, 12, 92, or 350. In someembodiments, the engineered polypeptide having imine reductase activitydisclosed herein is capable of converting the ketone substrate ofcompound (1j) and the amine substrate of compound (2g) to the amineproduct compound (3q) with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold,4 fold, 5 fold, 10 fold or more activity relative to the activity of thereference polypeptide of SEQ ID NO:6, 12, 146, or 198. In someembodiments, the engineered polypeptide having imine reductase activitydisclosed herein is capable of converting the ketone substrate ofcompound (1i) and the amine substrate of compound (2h) to the amineproduct compound (3r) with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold,4 fold, 5 fold, 10 fold or more activity relative to the activity of thereference polypeptide of SEQ ID NO:6, 12, 84, or 228. In someembodiments, the engineered polypeptide having imine reductase activitydisclosed herein is capable of converting the ketone substrate ofcompound (1e) and the amine substrate of compound (2d) to the amineproduct compound (3s) with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold,4 fold, 5 fold, 10 fold or more activity relative to the activity of thereference polypeptide of SEQ ID NO:6, 12, 162, or 354.

In addition to the positions of residue differences specified above, anyof the engineered imine reductase polypeptides disclosed herein canfurther comprise other residue differences relative to SEQ ID NO:6 atother residue positions than those of amino acid differences disclosedin Tables 3A-3L, i.e., residue positions other than X12, X18, X20, X26,X27, X29, X37, X57, X65, X74, X82, X87, X93, X94, X96, X108, X111, X126,X138, X140, X141, X142, X143, X153, X154, X156, X157, X158, X159, X163,X170, X175, X177, X195, X197, X200, X201, X220, X221, X223, X234, X241,X242, X253, X254, X256, X257, X259, X260, X261, X262, X263, X264, X265,X267, X270, X272, X273, X274, X276, X277, X278, X279, X281, X282, X283,X284, X291, X292, X295, X296, X326, and X352. Residue differences atthese other residue positions can provide for additional variations inthe amino acid sequence without adversely affecting the ability of thepolypeptide to catalyze one or more of the above conversion reactions(a)-(s) from Table 2. Accordingly, in some embodiments, in addition tothe amino acid residue differences present in any one of the engineeredimine reductase polypeptides selected from SEQ ID NOS:8-924, thesequence can further comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9,1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30,1-35, 1-40, 1-45, or 1-50 residue differences at other amino acidresidue positions as compared to the SEQ ID NO:6. In some embodiments,the number of amino acid residue differences as compared to thereference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45 or 50residue positions. In some embodiments, the number of amino acid residuedifferences as compared to the reference sequence can be 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25residue positions. The residue difference at these other positions canbe conservative changes or non-conservative changes. In someembodiments, the residue differences can comprise conservativesubstitutions and non-conservative substitutions as compared to thenaturally occurring imine reductase polypeptide of SEQ ID NO:2 or theengineered imine reductase polypeptide of SEQ ID NO:6.

In some embodiments, the present invention also provides engineeredpolypeptides that comprise a fragment of any of the engineered iminereductase polypeptides described herein that retains the functionalactivity and/or improved property of that engineered imine reductase.Accordingly, in some embodiments, the present invention provides apolypeptide fragment capable of catalyzing one or more of the aboveconversion reactions (a)-(s) of Table 2, under suitable reactionconditions, wherein the fragment comprises at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% of a full-length amino acid sequence of anengineered imine reductase polypeptide of the present invention, such asan exemplary engineered imine reductase polypeptide selected fromeven-numbered sequence identifiers SEQ ID NOS:8-924.

In some embodiments, the engineered imine reductase polypeptide can havean amino acid sequence comprising a deletion of any one of theengineered imine reductase polypeptides described herein, such as theexemplary engineered polypeptides of even-numbered sequence identifiersSEQ ID NO:8-924. Thus, for each and every embodiment of the engineeredimine reductase polypeptides of the invention, the amino acid sequencecan comprise deletions of one or more amino acids, 2 or more aminoacids, 3 or more amino acids, 4 or more amino acids, 5 or more aminoacids, 6 or more amino acids, 8 or more amino acids, 10 or more aminoacids, 15 or more amino acids, or 20 or more amino acids, up to 10% ofthe total number of amino acids, up to 10% of the total number of aminoacids, up to 20% of the total number of amino acids, or up to 30% of thetotal number of amino acids of the imine reductase polypeptides, wherethe associated functional activity and/or improved properties of theengineered imine reductase described herein is maintained. In someembodiments, the deletions can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35,1-40, 1-45, or 1-50 amino acid residues. In some embodiments, the numberof deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 aminoacid residues. In some embodiments, the deletions can comprise deletionsof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21,22, 23, 24, or 25 amino acid residues.

In some embodiments, the engineered imine reductase polypeptide hereincan have an amino acid sequence comprising an insertion as compared toany one of the engineered imine reductase polypeptides described herein,such as the exemplary engineered polypeptides of even-numbered sequenceidentifiers SEQ ID NOS: 8-924. Thus, for each and every embodiment ofthe imine reductase polypeptides of the invention, the insertions cancomprise one or more amino acids, 2 or more amino acids, 3 or more aminoacids, 4 or more amino acids, 5 or more amino acids, 6 or more aminoacids, 8 or more amino acids, 10 or more amino acids, 15 or more aminoacids, 20 or more amino acids, 30 or more amino acids, 40 or more aminoacids, or 50 or more amino acids, where the associated functionalactivity and/or improved properties of the engineered imine reductasedescribed herein is maintained. The insertions can be to amino orcarboxy terminus, or internal portions of the imine reductasepolypeptide.

In some embodiments, the engineered imine reductase polypeptide hereincan have an amino acid sequence comprising a sequence selected fromeven-numbered sequence identifiers SEQ ID NOS: 8-924, and optionally oneor several (e.g., up to 3, 4, 5, or up to 10) amino acid residuedeletions, insertions and/or substitutions. In some embodiments, theamino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40,1-45, or 1-50 amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the number of amino acid sequencehas optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residuedeletions, insertions and/or substitutions. In some embodiments, thesubstitutions can be conservative or non-conservative substitutions.

In the above embodiments, the suitable reaction conditions for theengineered polypeptides can be those HTP assay conditions described inTables 3A-3L and the Examples. Guidance for use of these foregoing HTPand SFP reaction conditions and the imine reductase polypeptides aregiven in, among others, Tables 3A-3L, and the Examples.

In some embodiments, the polypeptides of the invention can be in theform of fusion polypeptides in which the engineered polypeptides arefused to other polypeptides, such as, by way of example and notlimitation, antibody tags (e.g., myc epitope), purification sequences(e.g., His tags for binding to metals), and cell localization signals(e.g., secretion signals). Thus, the engineered polypeptides describedherein can be used with or without fusions to other polypeptides.

It is to be understood that the polypeptides described herein are notrestricted to the genetically encoded amino acids. In addition to thegenetically encoded amino acids, the polypeptides described herein maybe comprised, either in whole or in part, of naturally-occurring and/orsynthetic non-encoded amino acids. Certain commonly encounterednon-encoded amino acids of which the polypeptides described herein maybe comprised include, but are not limited to: the D-stereomers of thegenetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr);α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine(Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug);N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine(Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Nat); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mct);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Pat); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

In some embodiments, the engineered polypeptides can be in variousforms, for example, such as an isolated preparation, as a substantiallypurified enzyme, whole cells transformed with gene(s) encoding theenzyme, and/or as cell extracts and/or lysates of such cells. Theenzymes can be lyophilized, spray-dried, precipitated or be in the formof a crude paste, as further discussed below.

In some embodiments, the engineered polypeptides can be provided on asolid support, such as a membrane, resin, solid carrier, or other solidphase material. A solid support can be composed of organic polymers suchas polystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. The configuration of a solid support can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression, or other container, vessel, feature, orlocation.

In some embodiments, the engineered polypeptides having imine reductaseactivity of the present invention can be immobilized on a solid supportsuch that they retain their improved activity, stereoselectivity, and/orother improved properties relative to the reference engineeredpolypeptide of SEQ ID NO:6. In such embodiments, the immobilizedpolypeptides can facilitate the biocatalytic conversion of the ketoneand amine substrate compounds of formula (I) and formula (II) to theamine product compound of formula (III), (e.g., as in conversionreactions (a)-(s) of Table 2), and after the reaction is complete areeasily retained (e.g., by retaining beads on which polypeptide isimmobilized) and then reused or recycled in subsequent reactions. Suchimmobilized enzyme processes allow for further efficiency and costreduction. Accordingly, it is further contemplated that any of themethods of using the imine reductase polypeptides of the presentinvention can be carried out using the same imine reductase polypeptidesbound or immobilized on a solid support.

Methods of enzyme immobilization are well-known in the art. Theengineered polypeptides can be bound non-covalently or covalently.Various methods for conjugation and immobilization of enzymes to solidsupports (e.g., resins, membranes, beads, glass, etc.) are well known inthe art (See e.g., Yi et al., Proc. Biochem., 42: 895-898 [2007]; Martinet al., Appl. Micro. and Biotech., 76: 843-851 [2007]; Koszelewski etal., J. Mol. Cat. B: Enzy., 63: 39-44 [2010]; Truppo et al., Org. Proc.Res. Dev., published online dx.doi.org/10.1021/op200157c; Hermanson,Bioconjugate Techniques, Second Edition, Academic Press [2008]; Mateo etal., Biotech. Prog., 18:629-34 [2002]; and C. M. Niemeyer [ed.],Bioconjugation Protocols: Strategies and Methods, In Methods inMolecular Biology, Humana Press [2004], each of which are incorporatedby reference herein). Solid supports useful for immobilizing theengineered imine reductases of the present invention include but are notlimited to beads or resins comprising polymethacrylate with epoxidefunctional groups, polymethacrylate with amino epoxide functionalgroups, styrene/DVB copolymer or polymethacrylate with octadecylfunctional groups. Exemplary solid supports useful for immobilizing theengineered imine reductase polypeptides of the present inventioninclude, but are not limited to, chitosan beads, Eupergit C, andSEPABEADs (Mitsubishi), including the following different types ofSEPABEAD: EC-EP, EC-HFA/S, EXA252, EXE119 and EXE120.

In some embodiments, the polypeptides described herein can be providedin the form of kits. The enzymes in the kits may be present individuallyor as a plurality of enzymes. The kits can further include reagents forcarrying out the enzymatic reactions, substrates for assessing theactivity of enzymes, as well as reagents for detecting the products. Thekits can also include reagent dispensers and instructions for use of thekits.

In some embodiments, the kits of the present invention include arrayscomprising a plurality of different imine reductase polypeptides atdifferent addressable position, wherein the different polypeptides aredifferent variants of a reference sequence each having at least onedifferent improved enzyme property. In some embodiments, a plurality ofpolypeptides immobilized on solid supports can be configured on an arrayat various locations, addressable for robotic delivery of reagents, orby detection methods and/or instruments. The array can be used to test avariety of substrate compounds for conversion by the polypeptides. Sucharrays comprising a plurality of engineered polypeptides and methods oftheir use are known in the art (See e.g., WO2009/008908A2).

6.4 Polynucleotides Encoding Engineered Imine Reductases, ExpressionVectors and Host Cells

In another aspect, the present invention provides polynucleotidesencoding the engineered imine reductase polypeptides described herein.The polynucleotides may be operatively linked to one or moreheterologous regulatory sequences that control gene expression to createa recombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered imine reductase can be introduced into appropriate hostcells to express the corresponding imine reductase polypeptide.

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode the improved imine reductase enzymes. Thus, havingknowledge of a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids by simply modifying thesequence of one or more codons in a way which does not change the aminoacid sequence of the protein. In this regard, the present inventionspecifically contemplates each and every possible variation ofpolynucleotides that could be made encoding the polypeptides describedherein by selecting combinations based on the possible codon choices,and all such variations are to be considered specifically disclosed forany polypeptide described herein, including the amino acid sequencespresented in Tables 3A-3L and disclosed in the sequence listingincorporated by reference herein as even-numbered sequence identifiersSEQ ID NOS:8-924.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells. In some embodiments, all codons need not be replaced to optimizethe codon usage of the imine reductases since the natural sequence willcomprise preferred codons and because use of preferred codons may not berequired for all amino acid residues. Consequently, codon optimizedpolynucleotides encoding the imine reductase enzymes may containpreferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90%of codon positions of the full length coding region.

In some embodiments, the polynucleotide comprises a codon optimizednucleotide sequence encoding the naturally occurring imine reductasepolypeptide of SEQ ID NO:2. In some embodiments, the polynucleotide hasa nucleic acid sequence comprising at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identity to the codon optimized nucleicacid sequence of SEQ ID NO:1. The codon optimized sequence of SEQ IDNO:1 enhances expression of the encoded, naturally occurring iminereductase.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference sequence of SEQ IDNO:1, or a complement thereof, and encodes a polypeptide having iminereductase activity.

In some embodiments, as described above, the polynucleotide encodes anengineered polypeptide having imine reductase activity with improvedproperties as compared to SEQ ID NO:6, where the polypeptide comprisesan amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to areference sequence selected from even-numbered sequence identifiers SEQID NOS:8-924, and at least one of the following features:

(i) a residue difference as compared to the reference sequence of SEQ IDNO:6 at a position selected from X12, X18, X26, X27, X57, X65, X87, X93,X96, X126, X138, X140, X142, X159, X170, X175, X177, X195, X200, X221,X234, X241, X242, X253, X254, X257, X262, X263, X267, X272, X276, X277,X278, X281, X282, X291, and X352, optionally wherein the residuedifference at the position is selected from X12M, X18G, X26M/V, X27S,X57D/L/V, X65I/V, X87A, X93G/Y, X96C, X126S, X138L, X140M, X142A,X159C/L/Q/V, X170F/K/R/S, X175R, X177R, X195S, X200S, X221F, 234C/L,X241K, X242C/L, X253K/N, X254R, X257Q, X262F/G/P/V,X263C/D/E/H/M/L/M/N/P/Q/V, X267E/G/H/I/N/S, X272D, X276L, X277H/L,X278E/H/K/N/R/S/W, X281A, X282A/R, X291E, and X352Q;

(ii) a residue difference as compared to the reference sequence of SEQID NO:6 selected from X20V, X29K, X37P, X74W, X82C/T, X94N, X108S,X111A/H, X141M/N, X143F/L/Y, X153F, X154C/D/G/K/L/N/S/T/V,X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V, X163V, X197V, X201I,X220C/K/Q, X223S, X256A/E/I/L/S/T, X259C/R, X260A/D/N/Q/V/Y,X261E/F/H/L/P/Q/Y, X264V, X270L, X273C, X274L/S, X279T, X284C/F/H/P/Q/S,X292E/P, and X295F; and/or

(iii) two or more residue differences as compared to the referencesequence of SEQ ID NO:6 selected from X82P, X141W, X153Y, X154F,X259I/L/M, X274L/M, X283V, and X296N/V. In some embodiments, thereference sequence is selected from SEQ ID NOS:6, 12, 84, 92, 146, 162,198, 228, 250, 354, and 440.

In some embodiments, the polynucleotide encodes a imine reductasepolypeptide capable of converting substrate compounds (1j) and (2b) tothe product compound (3o) with improved properties as compared to SEQ IDNO:6, wherein the polypeptide comprises an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to reference sequence SEQ IDNO:6 and one or more residue differences as compared to SEQ ID NO:6 atresidue positions selected from: X82P, X141W, X143W, X153Y, X154F/Q/Y,X256V, X259I/L/M/T, X260G, X261R, X265L, X273W, X274M, X277A/I, X279L,X283V, X284L, X296N, X326V.

In some embodiments, the polynucleotide encodes a imine reductasepolypeptide capable of converting substrate compounds (1j) and (2b) tothe product compound (3o) with improved properties as compared to SEQ IDNO:6, wherein the polypeptide comprises an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to reference sequence SEQ IDNO:6, and at least a combination of residue differences as compared toSEQ ID NO:6 selected from: (a) X153Y, and X283V; (b) X141W, X153Y, andX283V; (c) X141W, X153Y, X274L/M, and X283V; (d) X141W, X153Y, X154F,X274L/M, and X283V; (e) X141W, X153Y, X154F, and X283V; (f) X141W,X153Y, X283V, and X296N/V; (g) X141W, X153Y, X274L/M, X283V, andX296N/V; (h) X111A, X153Y, X256E, X274M, and X283V; (i) X111A, X141W,X153Y, X273C, X274M, X283V, and X284S; (j) X111A, X141W, X153Y, X273C,and X283V; (k) X111A, X141W, X153Y, X154F, X256E, X274M, X283V, X284S,and X296N; (l) X111A, X141W, X153Y, X256E, X273W, X274L, X283V, X284S,and X296N; (m) X111H, X141W, X153Y, X273W, X274M, X284S, and X296N; (n)X111H, X141W, X153Y, X154F, X273W, X274L, X283V, X284S, and X296N; (o)X82P, X141W, X153Y, X256E, X274M, and X283V; (p) X82P, X111A, X141W,X153Y, X256E, X274M, X283V, M284S, and E296V; (q) X94N, X143W, X159L,X163V, X259M, and X279L; (r) X141W, X153Y, X154F, and X256E; and (s)X153Y, X256E, and X274M.

In some embodiments, the polynucleotide encodes an engineered iminereductase polypeptide capable of converting substrate compounds (1j) and(2b) to the product compound (3o) with improved enzyme properties ascompared to the reference polypeptide of SEQ ID NO:2, wherein thepolypeptide comprises an amino acid sequence having at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to a reference polypeptide selected from any one ofeven-numbered sequence identifiers SEQ ID NOS:8-924, with the provisothat the amino acid sequence comprises any one of the set of residuedifferences as compared to SEQ ID NO:6 contained in any one of thepolypeptide sequences of even-numbered sequence identifiers SEQ IDNOS:8-924, as listed in Tables 3A-3L.

In some embodiments, the polynucleotide encoding the engineered iminereductase comprises an polynucleotide sequence selected from theodd-numbered sequence identifiers SEQ ID NOS:7-603.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from the odd-numbered sequence identifiers SEQ ID NOS:7-603, ora complement thereof, and encodes a polypeptide having imine reductaseactivity with one or more of the improved properties described herein.In some embodiments, the polynucleotide capable of hybridizing underhighly stringent conditions encodes a imine reductase polypeptide thathas an amino acid sequence that comprises one or more residuedifferences as compared to SEQ ID NO:6 at residue positions selectedfrom: X12, X18, X20, X26, X27, X29, X37, X57, X65, X74, X82, X87, X93,X94, X96, X108, X111, X126, X138, X140, X141, X142, X143, X153, X154,X156, X157, X158, X159, X163, X170, X175, X177, X195, X197, X200, X201,X220, X221, X223, X234, X241, X242, X253, X254, X256, X257, X259, X260,X261, X262, X263, X264, X265, X267, X270, X272, X273, X274, X276, X277,X278, X279, X281, X282, X283, X284, X291, X292, X295, X296, X326, andX352. In some embodiments, the specific residue differences at theseresidue positions are selected from: X12M, X18G, X20V, X26M/V, X27S,X29K, X37P, X57D/L/V, X65I/V, X74W, X82C/P/T, X87A, X93G/Y, X94N, X96C,X108S, X111A/H, X126S, X138L, X140M, X141M/N/W, X142A, X143F/L/W/Y,X153E/F/Y, X154C/D/F/G/K/L/N/Q/S/T/V/Y, X156H/L/N/M/R, X157F/Q/T/Y,X158I/L/R/S/T/V, X159C/L/Q/V, X163V, X170F/K/R/S, X175R, X177R, X195S,X197V, X200S, X201I, X220C/K/Q, X221F, X223S, X234V/C/L, X241K, X242C/L,X253K/N, X254R, X256A/E/I/L/S/T/V, X257Q, X259C/I/L/M/R/T,X260A/D/G/N/Q/V/Y, X261E/F/H/L/P/Q/R/Y, X262F/G/P/V,X263C/D/E/H/M/L/M/N/P/Q/V, X264V, X265L, X267E/G/H/I/N/S, X270L, X272D,X273C/W, X274L/M/S, X276L, X277A/H/I/L, X278E/H/K/N/R/S/W, X279L/T,X281A, X282A/R, X283M/V, X284C/F/H/L/P/Q/S, X291E, X292E/P, X295F,X296N, X326V, and X352Q.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity at the nucleotide level to areference polynucleotide encoding the engineered imine reductase. Insome embodiments, the reference polynucleotide sequence is selected fromthe odd-numbered sequence identifiers SEQ ID NOS:7-603.

An isolated polynucleotide encoding an improved imine reductasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. In some embodiments, the polynucleotidesencoding the polypeptides can be provided as expression vectors whereone or more control sequences is present to regulate the expression ofthe polynucleotides and/or polypeptides. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3^(rd)Ed., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2006.

In some embodiments, the control sequences include among others,promoters, leader sequence, polyadenylation sequence, propeptidesequence, signal peptide sequence, and transcription terminator.Suitable promoters can be selected based on the host cells used. Forbacterial host cells, suitable promoters for directing transcription ofthe nucleic acid constructs of the present invention, include thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (Villa-Kamaroff et al., Proc. Natl Acad.Sci. USA 75: 3727-3731 [1978]), as well as the tac promoter (DeBoer etal., Proc. Natl Acad. Sci. USA 80: 21-25 [1983]). Exemplary promotersfor filamentous fungal host cells, include promoters obtained from thegenes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei asparticproteinase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-likeprotease (See e.g., WO 96/00787), as well as the NA2-tpi promoter (ahybrid of the promoters from the genes for Aspergillus niger neutralalpha-amylase and Aspergillus oryzae triose phosphate isomerase), andmutant, truncated, and hybrid promoters thereof. Exemplary yeast cellpromoters can be from the genes can be from the genes for Saccharomycescerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase(GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are known in the art (See e.g., Romanoset al., Yeast 8:423-488 [1992]).

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention. For example, exemplary transcription terminatorsfor filamentous fungal host cells can be obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease.Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are also known in the art (See e.g., Romanos et al.,supra).

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are known in the art (Seee.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990 [1995]).

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. Any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention. Effectivesignal peptide coding regions for bacterial host cells are the signalpeptide coding regions obtained from the genes for Bacillus NC1B 11837maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are known in the art(See e.g., Simonen and Palva, Microbiol. Rev., 57: 109-137 [1993]).Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase. Usefulsignal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are known in theart (See e.g., Romanos et al., supra).

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila lactase (See e.g., WO95/33836). Where both signal peptide and propeptide regions are presentat the amino terminus of a polypeptide, the propeptide region ispositioned next to the amino terminus of a polypeptide and the signalpeptide region is positioned next to the amino terminus of thepropeptide region.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide of thepresent invention would be operably linked with the regulatory sequence.

In another aspect, the present invention is also directed to arecombinant expression vector comprising a polynucleotide encoding anengineered imine reductase polypeptide, and one or more expressionregulating regions such as a promoter and a terminator, a replicationorigin, etc., depending on the type of hosts into which they are to beintroduced. The various nucleic acid and control sequences describedabove may be joined together to produce a recombinant expression vectorwhich may include one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding thepolypeptide at such sites. Alternatively, the nucleic acid sequence ofthe present invention may be expressed by inserting the nucleic acidsequence or a nucleic acid construct comprising the sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector (i.e., avector that exists as an extrachromosomal entity), the replication ofwhich is independent of chromosomal replication (e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificialchromosome). The vector may contain any means for assuringself-replication. Alternatively, the vector may be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. Furthermore, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon may beused.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol (Example 1) or tetracycline resistance.Suitable markers for yeast host cells include, but are not limited toADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for usein a filamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

In another aspect, the present invention provides a host cell comprisinga polynucleotide encoding an improved imine reductase polypeptide of thepresent invention, the polynucleotide being operatively linked to one ormore control sequences for expression of the imine reductase enzyme inthe host cell. Host cells for use in expressing the polypeptides encodedby the expression vectors of the present invention are well known in theart and include but are not limited to, bacterial cells, such as E.coli, Bacillus subtilis, Streptomyces and Salmonella typhimurium cells;fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae orPichia pastoris (ATCC Accession No. 201178)); insect cells such asDrosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary hostcells are Escherichia coli W3110 (AfhuA) and BL21.

Appropriate culture mediums and growth conditions for theabove-described host cells are well known in the art. Polynucleotidesfor expression of the imine reductase may be introduced into cells byvarious methods known in the art. Techniques include among others,electroporation, biolistic particle bombardment, liposome mediatedtransfection, calcium chloride transfection, and protoplast fusion.

In some embodiments, the polypeptides can be expressed in cell freeexpression systems (See e.g., Kudlicki et al., Cell Free Expression,1^(st) Ed., Landes Biosciences [2007]; and Spirin et al. (eds.), CellFree Protein Synthesis: Methods and Protocols, 1^(st) ed., Wiley-VCH[2007], all of which are incorporated herein by reference).

In the embodiments herein, the improved polypeptides and correspondingpolynucleotides can be obtained using methods used by those skilled inthe art. The engineered imine reductases described herein can beobtained by subjecting the polynucleotide encoding the naturallyoccurring gene encoding the wild-type opine dehydrogenase CENDH (SEQ IDNO:2) or another engineered imine reductase to mutagenesis and/ordirected evolution methods, as discussed above.

For example, mutagenesis and directed evolution methods can be readilyapplied to polynucleotides to generate variant libraries that can beexpressed, screened, and assayed. Mutagenesis and directed evolutionmethods are well known in the art (See e.g., U.S. Pat. Nos. 5,605,793,5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548,6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,277,638,6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883,6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198,6,344,356, 6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377,6,365,408, 6,368,861, 6,372,497, 6,376,246, 6,379,964, 6,387,702,6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745,6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468,6,455,253, 6,479,652, 6,482,647, 6,489,146, 6,506,602, 6,506,603,6,519,065, 6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467,6,579,678, 6,586,182, 6,602,986, 6,613,514, 6,653,072, 6,716,631,6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297,7,148,054, 7,288,375, 7,421,347, 7,430,477, 7,534,564, 7,620,500,7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986,7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,873,499,7,904,249, and 7,957,912, and all related non-US counterparts; Ling etal., Anal. Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol.Biol., 57:369-74 [1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985];Botstein et al., Science, 229:1193-1201 [1985]; Carter, Biochem. J.,237:1-7 [1986]; Kramer et al., Cell, 38:879-887 [1984]; Wells et al.,Gene, 34:315-323 [1985]; Minshull et al., Curr. Op. Chem. Biol.,3:284-290 [1999]; Christians et al., Nat. Biotechnol., 17:259-264[1999]; Crameri et al., Nature, 391:288-291 [1998]; Crameri, et al.,Nat. Biotechnol., 15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci.U.S.A., 94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol.,14:315-319 [1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc.Nat. Acad. Sci. USA, 91:10747-10751 [1994]; US Pat. Appln. Publn. Nos.2008/0220990, and US 2009/0312196; WO 95/22625, WO 97/0078, WO 97/35966,WO 98/27230, WO 00/42651, WO 01/75767, and WO 2009/152336; all of whichare incorporated herein by reference). Other directed evolutionprocedures that find use include, but are not limited to can be usedinclude, among others, staggered extension process (StEP), in vitrorecombination (See e.g., Zhao et al., Nat. Biotechnol., 16:258-261[1998]), mutagenic PCR (See e.g., Caldwell et al., PCR Meth. Appl.,3:S136-S140 [1994]), and cassette mutagenesis (See e.g., Black et al.,Proc. Natl. Acad. Sci. USA 93:3525-3529 [1996]), all of which areincorporated herein by reference.

The clones obtained following mutagenesis treatment can be screened forengineered imine reductases having one or more desired improved enzymeproperties. For example, where the improved enzyme property desired isincrease activity in the conversion of a ketone of compound (1b) and anamine of compound (2b) to a secondary amine of compound (3d), enzymeactivity may be measured for production of compound (3d). Clonescontaining a polynucleotide encoding a imine reductase with the desiredcharacteristics, e.g., increased production of compound (3d), are thenisolated, sequenced to identify the nucleotide sequence changes (ifany), and used to express the enzyme in a host cell. Measuring enzymeactivity from the expression libraries can be performed using thestandard biochemistry techniques, such as HPLC analysis and/orderivatization of products (pre or post separation), e.g., with dansylchloride or OPA.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides encodingportions of the imine reductase can be prepared by chemical synthesisusing the classical phosphoramidite method (See e.g., Beaucage et al.,Tetra. Lett., 22:1859-69 [1981]), or alternative methods (See e.g.,Matthes et al., EMBO J., 3:801-05 [1984]), as it is typically practicedin automated synthetic methods. According to the phosphoramidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,purified, annealed, ligated and cloned in appropriate vectors. Inaddition, essentially any nucleic acid can be obtained from any of avariety of commercial sources. In some embodiments, additionalvariations can be created by synthesizing oligonucleotides containingdeletions, insertions, and/or substitutions, and combining theoligonucleotides in various permutations to create engineered iminereductases with improved properties.

Accordingly, in some embodiments, a method for preparing the engineeredimine reductases polypeptide can comprise: (a) synthesizing apolynucleotide encoding a polypeptide comprising an amino acid sequenceselected from the even-numbered sequence identifiers SEQ ID NO: 6-924,and having one or more residue differences as compared to SEQ ID NO:2 atresidue positions selected from: X12, X18, X20, X26, X27, X29, X37, X57,X65, X74, X82, X87, X93, X94, X96, X108, X111, X126, X138, X140, X141,X142, X143, X153, X154, X156, X157, X158, X159, X163, X170, X175, X177,X195, X197, X200, X201, X220, X221, X223, X234, X241, X242, X253, X254,X256, X257, X259, X260, X261, X262, X263, X264, X265, X267, X270, X272,X273, X274, X276, X277, X278, X279, X281, X282, X283, X284, X291, X292,X295, X296, X326, and X352; and (b) expressing the imine reductasepolypeptide encoded by the polynucleotide.

In some embodiments of the method, the residue differences at residuepositions X12, X18, X20, X26, X27, X29, X37, X57, X65, X74, X82, X87,X93, X94, X96, X108, X111, X126, X138, X140, X141, X142, X143, X153,X154, X156, X157, X158, X159, X163, X170, X175, X177, X195, X197, X200,X201, X220, X221, X223, X234, X241, X242, X253, X254, X256, X257, X259,X260, X261, X262, X263, X264, X265, X267, X270, X272, X273, X274, X276,X277, X278, X279, X281, X282, X283, X284, X291, X292, X295, X296, X326,and X352, are selected from X12M, X18G, X20V, X26M/V, X27S, X29K, X37P,X57D/L/V, X65I/V, X74W, X82C/P/T, X87A, X93G/Y, X94N, X96C, X108S,X111A/H, X126S, X138L, X140M, X141M/N/W, X142A, X143F/L/W/Y, X153E/F/Y,X154C/D/F/G/K/L/N/Q/S/TN/Y, X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V,X159C/L/Q/V, X163V, X170F/K/R/S, X175R, X177R, X195S, X197V, X200S,X201I, X220C/K/Q, X221F, X223S, X234V/C/L, X241K, X242C/L, X253K/N,X254R, X256A/E/I/L/S/T/V, X257Q, X259C/I/L/M/R/T, X260A/D/G/N/Q/V/Y,X261E/F/H/L/P/Q/R/Y, X262F/G/P/V, X263C/D/E/H/I/K/L/M/N/P/Q/V, X264V,X265L, X267E/G/H/I/N/S, X270L, X272D, X273C/W, X274L/M/S, X276L,X277A/H/I/L, X278E/H/K/N/R/S/W, X279L/T, X281A, X282A/R, X283M/V,X284C/F/H/L/P/Q/S, X291E, X292E/P, X295F, X296N, X326V, and X352Q.

In some embodiments of the method, the polynucleotide can encode anengineered imine reductase that has optionally one or several (e.g., upto 3, 4, 5, or up to 10) amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the number of amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,30, 35, 40, 45, or 50 amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the substitutions can beconservative or non-conservative substitutions.

In another aspect, the present invention provides methods ofmanufacturing the engineered imine reductase polypeptides, where themethod can comprise culturing a host cell capable of expressing apolynucleotide encoding the imine reductase polypeptide under conditionssuitable for expression of the polypeptide. The method can furthercomprise isolating or purifying the expressed imine reductasepolypeptide, as described herein.

In some embodiments, the method for preparing or manufacturing theengineered imine reductase polypeptides further comprises the step ofisolating the polypeptide. The engineered polypeptides can be expressedin appropriate cells, as described above, and isolated (or recovered)from the host cells, the culture medium, and/or expression medium usingany one or more of the well known techniques used for proteinpurification, including, among others, lysozyme treatment, sonication,filtration, salting-out, ultra-centrifugation, and chromatography.Suitable solutions for lysing and the high efficiency extraction ofproteins from bacteria, such as E. coli, are commercially available,such as CelLytic B™ from Sigma-Aldrich of St. Louis Mo. Chromatographictechniques for isolation of the imine reductase polypeptides include,among others, reverse phase chromatography high performance liquidchromatography, ion exchange chromatography, gel electrophoresis, andaffinity chromatography.

In some embodiments, the non-naturally occurring polypeptides of theinvention can be prepared and used in various forms including but notlimited to crude extracts (e.g., cell-free lysates), powders (e.g.,shake-flask powders), lyophilizates, and substantially pure preparations(e.g., DSP powders), as further illustrated in the Examples below.

In some embodiments, the engineered polypeptides can be prepared andused in purified form, for example a substantially purified form.Generally, conditions for purifying a particular polypeptide willdepend, in part, on factors such as net charge, hydrophobicity,hydrophilicity, molecular weight, molecular shape, etc., and will beapparent to those having skill in the art. To facilitate purification,it is contemplated that in some embodiments the engineered polypeptidescan be expressed as fusion proteins with purification tags, such asHis-tags having affinity for metals, or antibody tags for binding toantibodies, e.g., myc epitope tag.

In some embodiments, affinity techniques may be used to isolate theimproved imine reductase enzymes. For affinity chromatographypurification, any antibody which specifically binds the imine reductasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with a imine reductase polypeptide, or a fragmentthereof. The imine reductase polypeptide or fragment may be attached toa suitable carrier, such as BSA, by means of a side chain functionalgroup or linkers attached to a side chain functional group. In someembodiments, the affinity purification can use a specific ligand boundby the imine reductase, such as poly(L-proline) or dye affinity column(see, e.g., EP0641862; Stellwagen, E., 2001, “Dye AffinityChromatography,” In Current Protocols in Protein Science Unit9.2-9.2.16).

6.5 Methods of Using the Engineered Imine Reductase Enzymes

In another aspect, the engineered polypeptides having imine reductaseactivity described herein can be used in a process for converting acompound of formula (I) and a compound of formula (II) to a secondary ortertiary amine compound of formula (III) as described above andillustrated in Scheme 1. Generally, such a biocatalytic process forcarrying out the reductive amination reaction of Scheme 1 comprisescontacting or incubating the ketone and amine substrate compounds withan engineered polypeptide having imine reductase activity of the presentinvention in the presence of a cofactor, such as NADH or NADPH, underreaction conditions suitable for formation of the amine product compoundof formula (III).

In some embodiments, the imine reductases can be used in a process forpreparing a secondary or tertiary amine product compound of formula(III),

wherein, R¹ and R² groups are independently selected from optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl; and optionally R¹ andR² are linked to form a 3-membered to 10-membered ring; R³ and R⁴ groupsare independently selected from a hydrogen atom, and optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl, with the proviso thatboth R³ and R⁴ cannot be hydrogen; and optionally R³ and R⁴ are linkedto form a 3-membered to 10-membered ring; and optionally, the carbonatom and/or the nitrogen indicated by * is chiral. The process comprisescontacting a ketone compound of formula (I),

wherein R¹, and R² are as defined above; and an amine compound offormula (II),

wherein R³, and R⁴ are as defined above; with an engineered polypeptidehaving imine reductase activity in presence of a cofactor under suitablereaction conditions.

As illustrated by the reactions in Table 2, and Tables 3A-3L, theengineered polypeptides having imine reductase activity of the presentinvention have activity with, or can be further engineered to haveactivity with, a wide range of amine substrate compounds of formula (II)in a process for preparing compound of formula (III). Accordingly, insome embodiments of the above biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), thecompound of formula (II) can be a primary amine wherein at least one ofR³ and R⁴ is hydrogen, whereby the product of formula (III) is asecondary amine compound. In some embodiments of the process, neither R³or R⁴ is hydrogen and the compound of formula (II) is a secondary amine,whereby the compound of formula (III) is tertiary amine. In someembodiments of the process, the compound of formula (II) is a secondaryamine and R³ or R⁴ are different, whereby the nitrogen atom indicatedby * of the amine compound of formula (III) is chiral. Further, in someembodiments, one stereoisomer of the chiral amine compound of formula(III) is formed stereoselectively, and optionally formed highlystereoselectively (e.g., in at least about 85% stereomeric excess).

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R³and R⁴ groups of the compound of formula (II) are linked to form a3-membered to 10-membered ring. In some embodiments, the ring is a5-membered to 8-membered is an optionally substituted cycloalkyl, aryl,arylalkyl, heterocycloalkyl, heteroaryl, or heteroarylalkyl ring.

In some embodiments of the biocatalytic process for preparing an amineproduct compound of formula (III), the compound of formula (II) is aprimary amine, wherein R³ group is hydrogen, and R⁴ is selected fromoptionally substituted (C₁-C₆)alkyl, (C₁-C₆)alkenyl, (C₁-C₆)alkynyl,(C₁-C₆)carboxyalkyl, (C₁-C₆)aminoalkyl, (C₁-C₆)haloalkyl, and(C₁-C₆)alkylthioalkyl. In some embodiments, the R⁴ group is selectedfrom optionally substituted (C₁-C₆)alkyl, (C₁-C₆)carboxyalkyl, and(C₁-C₆)aminoalkyl. In some embodiments, the R⁴ group is optionallysubstituted (C₁-C₆)alkyl, or (C₁-C₆)carboxyalkyl. In some embodiments,the compound of formula (II) is selected from methylamine,dimethylamine, isopropylamine, butylamine, and isobutylamine. In someembodiments, the amine substrate compound R³ group is hydrogen, and R⁴is selected from optionally substituted (C₄-C₈)cycloalkyl,(C₄-C₈)heterocycloalkyl, (C₄-C₈)aryl, (C₄-C₈)arylalkyl,(C₄-C₈)heteroaryl, and (C₄-C₈)heteroarylalkyl. In some embodiments, theamine substrate compound R³ group is hydrogen, and R⁴ is selected fromoptionally substituted (C₄-C₈)aryl, (C₄-C₈)arylalkyl, (C₄-C₈)heteroaryl,and (C₄-C₈)heteroarylalkyl. In some embodiments, the amine substratecompound R³ group is hydrogen, and R⁴ is optionally substituted(C₄-C₈)aryl. In some embodiments, the compound of formula (II) isoptionally substituted aniline.

As illustrated by the reactions in Table 2, and Tables 3A-3L, theengineered polypeptides having imine reductase activity of the presentinvention have activity with, or can be further engineered to haveactivity with, a wide range of ketone substrate compounds of formula (I)in a process for preparing compound of formula (III). In someembodiments, the R¹ and R² groups of the ketone substrate of compound(I) are different, whereby the carbon atom indicated by * of the aminecompound of formula (III) is chiral. Further, in some embodiments of theprocess, one stereoisomer of the chiral amine compound of formula (III)is formed stereoselectively, and optionally formed highlystereoselectively (e.g., in at least about 85% stereomeric excess).

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹and R² groups of the compound of formula (I) are linked to form a3-membered to 10-membered ring. In some embodiments, the ring is anoptionally substituted cycloalkyl, aryl, arylalkyl, heterocycloalkyl,heteroaryl, or heteroarylalkyl ring. In some embodiments of the process,the compound of formula (I) is selected from optionally substitutedcyclobutanone, cyclopentanone, cyclohexanone, and cycloheptanone.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹and R² groups of the compound of formula (I) are independently selectedfrom optionally substituted (C₁-C₆)alkyl, (C₁-C₆)alkenyl,(C₁-C₆)alkynyl, (C₁-C₆)carboxyalkyl, (C₁-C₆)aminoalkyl,(C₁-C₆)haloalkyl, and (C₁-C₆)alkylthioalkyl.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹group of the compound of formula (I) is selected from optionallysubstituted (C₁-C₆)alkyl, (C₁-C₆)alkenyl, (C₁-C₆)alkynyl,(C₁-C₆)carboxyalkyl, (C₁-C₆)aminoalkyl, (C₁-C₆)haloalkyl, and(C₁-C₆)alkylthioalkyl; and the R² group of the compound of formula (I)is selected from optionally substituted (C₄-C₈)cycloalkyl,(C₄-C₈)heterocycloalkyl, (C₄-C₈)arylalkyl, (C₄-C₈)heteroaryl, and(C₄-C₈)heteroarylalkyl.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹group of the compound of formula (I) is carboxy. In some embodiments,the compound of formula (I) is a 2-keto-acid selected from pyruvic acid,2-oxo-propanoic acid, 2-oxo-butanoic acid, 2-oxo-pentanoic acid,2-oxo-hexanoic acid.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹group of the compound of formula (I) is a hydrogen atom, and thecompound of formula (I) is an aldehyde. In such embodiments, the R¹group of the compound of formula (I) is selected from optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl.

As illustrated by the compounds of formulas (I), (II), and (III), listedfor the reactions in Table 2, in some embodiments of the abovebiocatalytic process for preparing a secondary or tertiary amine productcompound of formula (III), the product compound of formula (III)comprises a compound selected from group consisting of: compound (3a),compound (3b), compound (3c), compound (3d), compound (3e), compound(3f), compound (3g), compound (3h), compound (3i), compound (3j),compound (3k), compound (3l), compound (3m), compound (3n), compound(3o), compound (3p), compound (3q), compound (3r), and compound (3s). Insome embodiments of the process, the compound of formula (I) comprises acompound selected from group consisting of: compound (1a), compound(1b), compound (1c), compound (1d), compound (1e), compound (1f),compound (1g), compound (1h), compound (1i), and compound (1j). In someembodiments of the process, the compound of formula (II) comprises acompound selected from group consisting of: compound (2a), compound(2b), compound (2c), compound (2d), compound (2e), compound (2f), andcompound (2g).

It is also contemplated that in some embodiments the process forpreparing an amine product compound of formula (III) catalyzed by anengineered polypeptide having imine reductase activity of the presentinvention comprises an intramolecular reaction, wherein the compound offormula (I) and the compound of formula (II) are groups on the same,single molecule. Thus, in some embodiments, at least one of R¹ and R² ofthe ketone compound of formula (I) is linked to at least one of R³ andR⁴ of the amine compound of formula (II), and the method comprisescontacting the single compound with a ketone group of formula (I) linkedto an amine group of formula (II) with an engineered polypeptide of thepresent invention under suitable reaction conditions. Illustrativeintramolecular reactions include but are not limited to reactions ofSchemes 2-5 shown below in Table 4, wherein groups R₁ and R₃ are asdefined above for groups R¹ and R³, and group R₅ is selected from ahydrogen atom, and optionally substituted alkyl, alkenyl, alkynyl,alkoxy, carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl,heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl,cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, andheteroarylalkyl.

TABLE 4 Scheme 2

Scheme 3

Scheme 4

Scheme 5

Without being bound by theory, it is believed that in most cases thebiocatalytic reaction of Scheme 1 involves the formation of anintermediate imine compound (e.g., an iminium intermediate) which isthen further reduced by the enzyme to the final secondary or tertiaryamine product compound of formula (III). It is also contemplated that insome embodiments, the process for preparing an amine product compound offormula (III) catalyzed by an engineered polypeptide having iminereductase activity of the present invention comprises contacting anengineered imine reductase polypeptide of the present invention with aketone compound of formula (I) and a primary amine compound of formula(II), whereby an imine intermediate is formed which then undergoes anintramolecular asymmetric cyclization reaction to yield a cyclicsecondary or tertiary hydroxyamine intermediate which undergoes hydroxylelimination to give a second imine (or enamine) intermediate. Thissecond imine (or enamine) is subsequently is then reduced in situ by theengineered imine reductase polypeptide of the present invention to yieldthe final cyclic amine product. Illustrative reactions involvingasymmetric cyclization through a hydroxyamine intermediate include butare not limited to reactions of Schemes 6-9 shown below in Table 5,wherein groups R₁ and R₃ are as defined above for groups R¹ and R³, andgroups R₅, R₆, and R₇ are independently selected from a hydrogen atom,and optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy,aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl,aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl.

TABLE 5 Scheme 6

Scheme 7

Scheme 8

Scheme 9

Without being bound by theory, it is believed that the engineeredpolypeptides having imine reductase activity (IREDs) mediate not onlythe formation of the imine and/or hydroxyamine intermiates as shown inthe reactions of Schemes 2-9 but also the conversion of the imineintermediates to the final amine product compound of formula (III)depicted by the second reaction arrow.

Generally, imine compounds are less stable than amine compounds andsusceptible to undesirable oxidation reactions. It is contemplated,however, that in some embodiments of the processes of the presentinvention, an imine compound, or an enamine compound which cantautomerize to form an imine, can form from a ketone of formula (I) andan amine compound of formula (II) absent the presence of an enzyme, andthen be contacted with an engineered polypeptide of the presentinvention to catalyze its conversion of the final to a secondary ortertiary amine product compound of formula (III). For example, an imineor enamine intermediate compound first can be formed by combining aketone of formula (I) and an amine compound of formula (II) as shown inSchemes 6-9 but without the presence of an IRED (i.e., an engineeredpolypeptide having imine reductase activity). The imine compound formeddirectly or from tautomerization of an enamine compound can then becontacted with an engineered polypeptide having imine reductase activityto catalyze the conversion to the final amine product compound offormula (III). In some embodiments, it is contemplated that the imine orenamine intermediate compound, where suitably stable, can be isolatedbefore carrying out a step of contacting it with an engineeredpolypeptide having imine reductase activity. Thus, it is contemplatedthat in some embodiments of the process, an imine or enamine compound isformed first from the compounds of formula (I) and formula (II), orthrough the intramolecular reaction of compound having a ketone grouplinked to an amine group, and then this imine or enamine compound iscontacted with an engineered polypeptide having imine reductase activityto form an amine product compound of formula (III).

In some embodiments, a stable imine or enamine compound may be obtained(i.e., without first reacting a ketone compound of formula (I) and anamine compound of formula (II)) and used directly as a substrate with anIRED. It is contemplated that in such embodiments, the biocatalyticprocess is carried out wherein there is only a single substrate which isa stable imine or enamine compound, and this compound is contacted withan engineered polypeptide having imine reductase activity of the presentinvention which catalyzes the reduction of the stable imine compound toform a secondary compound of formula (III). In such a reaction, thestereoselectivity of the engineered polypeptide can mediate theformation of a chiral center adjacent to the amine group of the compoundof formula (III). Table 6 (below) lists three examples of stable iminecompounds that can undergo chiral reduction in a biocatalytic processwith an engineered polypeptide of the present invention to produceintermediate compounds for the synthesis of the pharmaceuticalssolifenacin and tadalafil, and for the synthesis of the pharmaceuticalcompound, dexmethylphenidate.

TABLE 6 Imine or Enamine Product Compound Substrate Compound of formula(III)

Alternatively, it is also contemplated that any of the product compoundsof formula (III) produced via an IRED catalyzed reaction of an isolatedimine or enamine substrate compound (as shown in Table 6) could also beproduced via an IRED-catalyzed intramolecular reaction (like thoseillustrated in Table 4) using as substrate the open-chain version of theimine or enamine substrate compounds. Accordingly, each of the productcompounds of Table 6 could also be made using the intermolecularsubstrate shown in Table 7 with an engineered polypeptide having iminereductase activity of the present invention.

TABLE 7 Intermolecular Substrate Compound (comprising Product Compoundformula (I) and formula (II)) of formula (III)

There are numerous active pharmaceutical ingredient compounds thatinclude a secondary or tertiary amine group which could be produced viaa biocatalytic reductive amination using an engineered polypeptidehaving imine reductase activity of the present invention, and/or anengineered polypeptide produced by further directed evolution of anengineered polypeptide of the present invention. For example, Table 8lists various product compounds of formula (III) that are known activepharmaceutical ingredient compounds, or intermediate compounds usefulfor the synthesis of active pharmaceutical ingredient compounds, thatcould be produced using an engineered polypeptide having imine reductaseactivity of the present invention with the corresponding substratescompounds of formula (I) and/or formula (II).

TABLE 8 Substrate Compound Substrate Compound Product Compound offormula (I) of formula (II) of formula (III)

Me₂NH

Me₂NH

Me₂NH

In some embodiments of the above biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), theengineered polypeptide having imine reductase activity is derived from anaturally occurring opine dehydrogenase. In some embodiments, theengineered polypeptide having imine reductase activity is an engineeredpolypeptide derived from the opine dehydrogenase from Arthrobacter sp.strain 1C of SEQ ID NO:2, as disclosed herein, and exemplified by theengineered imine reductase polypeptides of even numbered sequenceidentifiers SEQ ID NOS:8-924.

Any of the engineered imine reductases described herein can be used inthe above biocatalytic processes for preparing a secondary or tertiaryamine compound of formula (III). By way of example and withoutlimitation, in some embodiments, the process can use an engineered iminereductase polypeptide comprising an amino acid sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identity to a reference sequence selected fromeven-numbered sequence identifiers SEQ ID NOS:4-924 and at least one ofthe following features:

(i) a residue difference as compared to the reference sequence of SEQ IDNO:6 at a position selected from X12, X18, X26, X27, X57, X65, X87, X93,X96, X126, X138, X140, X142, X159, X170, X175, X177, X195, X200, X221,X234, X241, X242, X253, X254, X257, X262, X263, X267, X272, X276, X277,X278, X281, X282, X291, and X352, optionally wherein the residuedifference at the position is selected from X12M, X18G, X26M/V, X27S,X57D/L/V, X65I/V, X87A, X93G/Y, X96C, X126S, X138L, X140M, X142A,X159C/L/Q/V, X170F/K/R/S, X175R, X177R, X195S, X200S, X221F, 234C/L,X241K, X242C/L, X253K/N, X254R, X257Q, X262F/G/P/V,X263C/D/E/H/I/K/L/M/N/P/Q/V, X267E/G/H/I/N/S, X272D, X276L, X277H/L,X278E/H/K/N/R/S/W, X281A, X282A/R, X291E, and X352Q;

(ii) a residue difference as compared to the reference sequence of SEQID NO:6 selected from X20V, X29K, X37P, X74W, X82C/T, X94N, X108S,X111A/H, X141M/N, X143F/L/Y, X153E/F, X154C/D/G/K/L/N/S/T/V,X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V, X163V, X197V, X201I,X220C/K/Q, X223S, X256A/E/I/L/S/T, X259C/R, X260A/D/N/Q/V/Y,X261E/F/H/L/P/Q/Y, X264V, X270L, X273C, X274L/S, X279T, X284C/F/H/P/Q/S,X292E/P, and X295F; and/or

(iii) two or more residue differences as compared to the referencesequence of SEQ ID NO:6 selected from X82P, X141W, X153Y, X154F,X259I/L/M, X274L/M, X283V, and X296N/V.

In some embodiments, the engineered polypeptide having imine reductaseactivity used in the above biocatalytic processes for preparing asecondary or tertiary amine compound of formula (III) comprises an aminoacid sequence comprising at least one residue difference as compared tothe reference sequence of SEQ ID NO: 6 selected from X12M, X37P, X82T,X111A, X154S, X156N/M, X223S, X256E, X260D, X261H, X262P, X263C/E/Q,X267G, X277L, X281A, X284P/S, and X292E. In some embodiments, amino acidsequence comprises at least one residue difference as compared to thereference sequence of SEQ ID NO: 6 selected from X256E, X93G/Y, X94N,X96C, X111A/H, X142A, X159L, X163V, X259R, X273C, and X284P/S. In someembodiments, the amino acid sequence comprises at least two residuedifferences as compared to the reference sequence of SEQ ID NO: 6selected from X82P, X141W, X143W, X153Y, X154F/Q/Y, X256V, X259I/L/M/T,X260G, X261R, X265L, X273W, X274M, X277A/I, X279L, X283V, X284L, X296N,X326V. In some embodiments, the at least two residue differences areselected from X141W, X153Y, X154F, X259I/L/M, X274L/M, X283V, andX296N/V.

In some embodiments, the engineered polypeptide having imine reductaseactivity used in the above biocatalytic processes for preparing asecondary or tertiary amine compound of formula (III) comprises an aminoacid sequence comprising at least a combination of residue differencesas compared to the reference sequence of SEQ ID NO:6 selected from: (a)X153Y, and X283V; (b) X141W, X153Y, and X283V; (c) X141W, X153Y,X274L/M, and X283V; (d) X141W, X153Y, X154F, X274L/M, and X283V; (e)X141W, X153Y, X154F, and X283V; (f) X141W, X153Y, X283V, and X296N/V;(g) X141W, X153Y, X274L/M, X283V, and X296N/V; (h) X111A, X153Y, X256E,X274M, and X283V; (i) X111A, X141W, X153Y, X273C, X274M, X283V, andX284S; (j) X111A, X141W, X153Y, X273C, and X283V; (k) X111A, X141W,X153Y, X154F, X256E, X274M, X283V, X284S, and X296N; (l) X111A, X141W,X153Y, X256E, X273W, X274L, X283V, X284S, and X296N; (m) X111H, X141W,X153Y, X273W, X274M, X284S, and X296N; (n) X111H, X141W, X153Y, X154F,X273W, X274L, X283V, X284S, and X296N; (o) X82P, X141W, X153Y, X256E,X274M, and X283V; (p) X82P, X111A, X141W, X153Y, X256E, X274M, X283V,M284S, and E296V; (q) X94N, X143W, X159L, X163V, X259M, and X279L; (r)X141W, X153Y, X154F, and X256E; and (s) X153Y, X256E, and X274M.

In some embodiments, the engineered polypeptide having imine reductaseactivity used in the above biocatalytic processes for preparing asecondary or tertiary amine compound of formula (III) comprises an aminoacid sequence comprising at least one of the above combinations of aminoacid residue differences (a)-(s), and further comprises at least oneresidue difference as compared to the reference sequence of SEQ ID NO:6selected from X12M, X18G, X20V, X26M/V, X27S, X29K, X37P, X57D/L/V,X65I/V, X74W, X82C/T, X87A, X93G/Y, X94N, X96C, X108S, X111A/H, X126S,X138L, X140M, X141M/N, X142A, X143F/L/Y, X153E/F, X154C/D/G/K/L/N/S/T/V,X156H/L/N/M/R, X157F/Q/T/Y, X158I/L/R/S/T/V, X159C/L/Q/V, X163V,X170F/K/R/S, X175R, X177R, X195S, X197V, X200S, X201I, X220C/K/Q, X221F,X223S, X234V/C/L, X241K, X242C/L, X253K/N, X254R, X256A/E/I/L/S/T,X257Q, X259C/R, X260A/D/N/Q/V/Y, X261E/F/H/L/P/Q/Y, X262P, X262F/G/V,X263C/D/E/H/I/K/L/M/N/P/Q/V, X264V, X267E/G/H/I/N/S, X270L, X272D,X273C, X274L/S, X276L, X277H/L, X278E/H/K/N/R/S/W, X279T, X281A,X282A/R, X284C/F/H/P/Q/S, X291E, X292E/P, X295F, and X352Q.

In some embodiments of the above processes, the exemplary iminereductases capable of carrying out the conversion reactions (a)-(s) ofTable 2 disclosed herein, can be used. This includes the engineeredpolypeptides disclosed herein comprising an amino acid sequence selectedfrom even-numbered sequence identifiers SEQ ID NOS:8-924. Guidance onthe choice and use of the engineered imine reductases is provided in thedescriptions herein, for example Tables 3A-3L, and the Examples.

In the embodiments herein and illustrated in the Examples, variousranges of suitable reaction conditions that can be used in theprocesses, include but are not limited to, substrate loading, cofactorloading, polypeptide loading, pH, temperature, buffer, solvent system,reaction time, and/or conditions with the polypeptide immobilized on asolid support. Further suitable reaction conditions for carrying out theprocess for biocatalytic conversion of substrate compounds to productcompounds using an engineered imine reductase polypeptide describedherein can be readily optimized in view of the guidance provided hereinby routine experimentation that includes, but is not limited to,contacting the engineered imine reductase polypeptide and ketone andamine substrate compounds of interest under experimental reactionconditions of concentration, pH, temperature, and solvent conditions,and detecting the product compound.

Generally, in the processes of the present invention, suitable reactionconditions include the presence of cofactor molecule which can act as anelectron donor in the reduction reaction carried out by the iminereductase. In some embodiments, the cofactor is selected from (but notlimited to) NADP⁺ (nicotinamide adenine dinucleotide phosphate), NADPH(the reduced form of NADP⁺), NAD (nicotinamide adenine dinucleotide) andNADH (the reduced form of NAD⁺). Generally, the reduced form of thecofactor is added to the enzyme reaction mixture. Accordingly, in someembodiments, the processes are carried out in presence of a cofactorselected from NADPH and NADH (these two cofactors are also referred toherein collectively as “NAD(P)H”). In some embodiments, the electrondonor is NADPH cofactor. In some embodiments, the process can be carriedout wherein the reaction conditions comprise an NADH or NADPH cofactorconcentration of about 0.03 to about 1 g/L, 0.03 to about 0.8 g/L, about0.03 to about 0.5 g/L, about 0.05 to about 0.3 g/L, about 0.05 to about0.2 g/L, or about 0.1 to about 0.2 g/L. In some embodiments, the processis carried out under NADH or NADPH cofactor concentration of about 1g/L, about 0.8 g/L, about 0.5 g/L, about 0.3 g/L, about 0.2 g/L, about0.1 g/L, about 0.05 g/L, or about 0.03 g/L.

In some embodiments of the process, an optional cofactor recyclingsystem, also referred to as a cofactor regeneration system, can be usedto regenerate cofactor NADPH/NADH from NADP+/NAD+ produced in theenzymatic reaction. A cofactor regeneration system refers to a set ofreactants that participate in a reaction that reduces the oxidized formof the cofactor (e.g., NADP⁺ to NADPH). Cofactors oxidized by thepolypeptide reduction of the keto substrate are regenerated in reducedform by the cofactor regeneration system. Cofactor regeneration systemscomprise a stoichiometric reductant that is a source of reducinghydrogen equivalents and is capable of reducing the oxidized form of thecofactor. The cofactor regeneration system may further comprise acatalyst, for example an enzyme catalyst, that catalyzes the reductionof the oxidized form of the cofactor by the reductant. Cofactorregeneration systems to regenerate NADH or NADPH from NAD⁺ or NADP⁺,respectively, are known in the art and can be used in the methodsdescribed herein.

Suitable exemplary cofactor regeneration systems that may be employed inthe imine reductase processes of the present invention include, but arenot limited to, formate and formate dehydrogenase, glucose and glucosedehydrogenase, glucose-6-phosphate and glucose-6-phosphatedehydrogenase, a secondary alcohol and alcohol dehydrogenase, phosphiteand phosphite dehydrogenase, molecular hydrogen and hydrogenase, and thelike. These systems may be used in combination with either NADP⁺/NADPHor NAD⁺/NADH as the cofactor. Electrochemical regeneration usinghydrogenase may also be used as a cofactor regeneration system (See,e.g., U.S. Pat. Nos. 5,538,867 and 6,495,023, both of which areincorporated herein by reference). Chemical cofactor regenerationsystems comprising a metal catalyst and a reducing agent (for example,molecular hydrogen or formate) may also be suitable (See, e.g., WO2000/053731, which is incorporated herein by reference).

In some embodiments, the co-factor regenerating system comprises aformate dehydrogenase, which is a NAD⁺ or NADP⁺-dependent enzyme thatcatalyzes the conversion of formate and NAD or NADP⁺ to carbon dioxideand NADH or NADPH, respectively. Formate dehydrogenases suitable for useas cofactor regenerating systems in the imine reductase processesdescribed herein include naturally occurring and non-naturally occurringformate dehydrogenases. Suitable formate dehydrogenases include, but arenot limited to those currently known in the art (See e.g., WO2005/018579, incorporated herein by reference). In some embodiments, theformate dehydrogenase used in the process is FDH-101, which commerciallyavailable (Codexis, Inc. Redwood City, Calif., USA). Formate may beprovided in the form of a salt, typically an alkali or ammonium salt(for example, HCO₂Na, KHCO₂NH₄, and the like), in the form of formicacid, typically aqueous formic acid, or mixtures thereof. A base orbuffer may be used to provide the desired pH.

In some embodiments, the cofactor recycling system comprises glucosedehydrogenase (GDH), which is a NAD⁺ or NADP⁺-dependent enzyme thatcatalyzes the conversion of D-glucose and NAD or NADP⁺ to gluconic acidand NADH or NADPH, respectively. Glucose dehydrogenases suitable for usein the practice of the imine reductase processes described hereininclude naturally occurring glucose dehydrogenases as well asnon-naturally occurring glucose dehydrogenases. Naturally occurringglucose dehydrogenase encoding genes have been reported in theliterature (e.g., the Bacillus subtilis 61297 GDH gene, B. cereus ATCC14579 and B. megaterium). Non-naturally occurring glucose dehydrogenasesare generated using any suitable method known in the art (e.g.,mutagenesis, directed evolution, and the like; See e.g., WO 2005/018579,and US Pat. Appln. Publ. Nos. 2005/0095619 and 2005/0153417, each ofwhich is incorporated by reference). In some embodiments, the glucosedehydrogenase used in the process is CDX-901 or GDH-105, each of whichcommercially available (Codexis, Inc. Redwood City, Calif., USA).

In some embodiments, the co-factor regenerating system comprises analcohol dehydrogenase or ketoreductase, which is an NAD⁺ orNADP⁺-dependent enzyme that catalyzes the conversion of a secondaryalcohol and NAD⁺ or NADP⁺ to a ketone and NADH or NADPH, respectively.Suitable secondary alcohols useful in cofactor regenerating systemsinclude lower secondary alkanols and aryl-alkyl carbinols, including butnot limited to, isopropanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol,3-pentanol, 3,3-dimethyl-2-butanol, and the like. Alcohol dehydrogenasessuitable for use as cofactor regenerating systems in the processesdescribed herein include naturally occurring and non-naturally occurringketoreductases. Naturally occurring alcohol dehydrogenase/ketoreductaseinclude known enzymes from, by way of example and not limitation,Thermoanerobium brockii, Rhodococcus erythropolis, Lactobacillus kefir,and Lactobacillus brevis, and non-naturally occurring alcoholdehydrogenases include engineered alcohol dehydrogenases derivedtherefrom. In some embodiments, non-naturally occurring ketoreductasesengineered for thermo- and solvent stability can be used. Suchketoreductases include those described herein, as well as others knownin the art (See e.g., US Pat. Appln. Publ. Nos. 20080318295A1, US20090093031A1, US 20090155863A1, US 20090162909A1, US 20090191605A1, US20100055751A1, WO/2010/025238A2, WO/2010/025287A2. and US 20100062499A1;each of which is incorporated by reference herein).

The concentration of the ketone and amine substrate compounds in thereaction mixtures can be varied, taking into consideration, for example,the desired amount of product compound, the effect of substrateconcentration on enzyme activity, stability of enzyme under reactionconditions, and the percent conversion of the substrates to the product.In some embodiments, the suitable reaction conditions comprise asubstrate compound loading of at least about 0.5 to about 200 g/L, 1 toabout 200 g/L, 5 to about 150 g/L, about 10 to about 100 g/L, 20 toabout 100 g/L or about 50 to about 100 g/L. In some embodiments, thesuitable reaction conditions comprise loading of each of the ketone andamine substrate compounds of at least about 0.5 g/L, at least about 1g/L, at least about 5 g/L, at least about 10 g/L, at least about 15 g/L,at least about 20 g/L, at least about 30 g/L, at least about 50 g/L, atleast about 75 g/L, at least about 100 g/L, at least about 150 g/L or atleast about 200 g/L, or even greater. The values for substrate loadingsprovided herein are based on the molecular weight of compound (1b),however it also contemplated that the equivalent molar amounts of otherketone and amine substrates, such as ketone substrate compounds(1a)-(1j), and amine substrate compounds (2a)-(2g), could be used, aswell as equimolar amounts of hydrates or salts of any of these compoundscan be used in the process. It is also contemplated that in someembodiments, the suitable reaction conditions comprise loading of eachof the ketone and amine substrate compounds in terms of molarconcentrations equivalent to the above g/L concentrations for compound(1b). Thus, reaction conditions can comprise a substrate loading of eachof the ketone and amine substrate compounds of at least about 5 mM, atleast about 10 mM, at least about 25 mM, at least about 50 mM, at leastabout 75 mM, at least about 100 mM, or even greater. In addition, it iscontemplated that substrate compounds covered by formulas (I) and (II),can be used in the same ranges of amounts as those used for compound(1b).

In carrying out the imine reductase mediated processes described herein,the engineered polypeptide may be added to the reaction mixture in theform of a purified enzyme, partially purified enzyme, whole cellstransformed with gene(s) encoding the enzyme, as cell extracts and/orlysates of such cells, and/or as an enzyme immobilized on a solidsupport. Whole cells transformed with gene(s) encoding the engineeredimine reductase enzyme or cell extracts, lysates thereof, and isolatedenzymes may be employed in a variety of different forms, including solid(e.g., lyophilized, spray-dried, and the like) or semisolid (e.g., acrude paste). The cell extracts or cell lysates may be partiallypurified by precipitation (ammonium sulfate, polyethyleneimine, heattreatment or the like, followed by a desalting procedure prior tolyophilization (e.g., ultrafiltration, dialysis, and the like). Any ofthe enzyme preparations (including whole cell preparations) may bestabilized by cross-linking using known cross-linking agents, such as,for example, glutaraldehyde or immobilization to a solid phase (e.g.,Eupergit C, and the like).

The gene(s) encoding the engineered imine reductase polypeptides can betransformed into host cell separately or together into the same hostcell. For example, in some embodiments one set of host cells can betransformed with gene(s) encoding one engineered imine reductasepolypeptide and another set can be transformed with gene(s) encodinganother engineered imine reductase polypeptide. Both sets of transformedcells can be utilized together in the reaction mixture in the form ofwhole cells, or in the form of lysates or extracts derived therefrom. Inother embodiments, a host cell can be transformed with gene(s) encodingmultiple engineered imine reductase polypeptide. In some embodiments theengineered polypeptides can be expressed in the form of secretedpolypeptides and the culture medium containing the secreted polypeptidescan be used for the imine reductase reaction.

The improved activity and/or stereoselectivity of the engineered iminereductase polypeptides disclosed herein provides for processes whereinhigher percentage conversion can be achieved with lower concentrationsof the engineered polypeptide. In some embodiments of the process, thesuitable reaction conditions comprise an engineered polypeptide amountof about 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 20% (w/w), 30% (w/w),40% (w/w), 50% (w/w), 75% (w/w), 100% (w/w) or more of substratecompound loading.

In some embodiments, the engineered polypeptides are present at about0.01 g/L to about 50 g/L; about 0.05 g/L to about 50 g/L; about 0.1 g/Lto about 40 g/L; about 1 g/L to about 40 g/L; about 2 g/L to about 40g/L; about 5 g/L to about 40 g/L; about 5 g/L to about 30 g/L; about 0.1g/L to about 10 g/L; about 0.5 g/L to about 10 g/L; about 1 g/L to about10 g/L; about 0.1 g/L to about 5 g/L; about 0.5 g/L to about 5 g/L; orabout 0.1 g/L to about 2 g/L. In some embodiments, the imine reductasepolypeptide is present at about 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/L,0.5 g/L, 1, 2 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35g/L, 40 g/L, or 50 g/L.

During the course of the reactions, the pH of the reaction mixture maychange. The pH of the reaction mixture may be maintained at a desired pHor within a desired pH range. This may be done by the addition of anacid or a base, before and/or during the course of the reaction.Alternatively, the pH may be controlled by using a buffer. Accordingly,in some embodiments, the reaction condition comprises a buffer. Suitablebuffers to maintain desired pH ranges are known in the art and include,by way of example and not limitation, borate, phosphate,2-(N-morpholino)ethanesulfonic acid (MES),3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine,and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), and the like. Insome embodiments, the buffer is phosphate. In some embodiments of theprocess, the suitable reaction conditions comprise a buffer (e.g.,phosphate) concentration is from about 0.01 to about 0.4 M, 0.05 toabout 0.4 M, 0.1 to about 0.3 M, or about 0.1 to about 0.2 M. In someembodiments, the reaction condition comprises a buffer (e.g., phosphate)concentration of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.07, 0.1, 0.12,0.14, 0.16, 0.18, 0.2, 0.3, or 0.4 M. In some embodiments, the reactionconditions comprise water as a suitable solvent with no buffer present.

In the embodiments of the process, the reaction conditions can comprisea suitable pH. The desired pH or desired pH range can be maintained byuse of an acid or base, an appropriate buffer, or a combination ofbuffering and acid or base addition. The pH of the reaction mixture canbe controlled before and/or during the course of the reaction. In someembodiments, the suitable reaction conditions comprise a solution pHfrom about 4 to about 10, pH from about 5 to about 10, pH from about 7to about 11, pH from about 8 to about 10, pH from about 6 to about 8. Insome embodiments, the reaction conditions comprise a solution pH ofabout 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

In the embodiments of the processes herein, a suitable temperature canbe used for the reaction conditions, for example, taking intoconsideration the increase in reaction rate at higher temperatures, andthe activity of the enzyme during the reaction time period. Accordingly,in some embodiments, the suitable reaction conditions comprise atemperature of from about 10° C. to about 80° C., about 10° C. to about70° C., about 15° C. to about 65° C., about 20° C. to about 60° C.,about 20° C. to about 55° C., about 25° C. to about 55° C., or about 30°C. to about 50° C. In some embodiments, the suitable reaction conditionscomprise a temperature of about 10° C., 15° C., 20° C., 25° C., 30° C.,35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C.,or 80° C. In some embodiments, the temperature during the enzymaticreaction can be maintained at a specific temperature throughout thecourse of the reaction. In some embodiments, the temperature during theenzymatic reaction can be adjusted over a temperature profile during thecourse of the reaction.

The processes of the invention are generally carried out in a solvent.Suitable solvents include water, aqueous buffer solutions, organicsolvents, polymeric solvents, and/or co-solvent systems, which generallycomprise aqueous solvents, organic solvents and/or polymeric solvents.The aqueous solvent (water or aqueous co-solvent system) may bepH-buffered or unbuffered. In some embodiments, the processes using theengineered imine reductase polypeptides can be carried out in an aqueousco-solvent system comprising an organic solvent (e.g., ethanol,isopropanol (IPA), dimethyl sulfoxide (DMSO), dimethylformamide (DMF),dimethylacetamide (DMAc), N-methyl-pyrrolidone (NMP), ethyl acetate,butyl acetate, 1-octanol, heptane, octane, methyl t butyl ether (MTBE),toluene, and the like), ionic or polar solvents (e.g.,1-ethyl-4-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium hexafluorophosphate, glycerol, polyethyleneglycol (PEG), and the like). In some embodiments, the co-solvent can bea polar solvent, such as a polyol, dimethylsulfoxide (DMSO), or loweralcohol. The non-aqueous co-solvent component of an aqueous co-solventsystem may be miscible with the aqueous component, providing a singleliquid phase, or may be partly miscible or immiscible with the aqueouscomponent, providing two liquid phases. Exemplary aqueous co-solventsystems can comprise water and one or more co-solvents selected from anorganic solvent, polar solvent, and polyol solvent. In general, theco-solvent component of an aqueous co-solvent system is chosen such thatit does not adversely inactivate the imine reductase enzyme under thereaction conditions. Appropriate co-solvent systems can be readilyidentified by measuring the enzymatic activity of the specifiedengineered imine reductase enzyme with a defined substrate of interestin the candidate solvent system, utilizing an enzyme activity assay,such as those described herein.

In some embodiments of the process, the suitable reaction conditionscomprise an aqueous co-solvent, where the co-solvent comprises DMSO atabout 1% to about 50% (v/v), about 1 to about 40% (v/v), about 2% toabout 40% (v/v), about 5% to about 30% (v/v), about 10% to about 30%(v/v), or about 10% to about 20% (v/v). In some embodiments of theprocess, the suitable reaction conditions can comprise an aqueousco-solvent comprising DMSO at about 1% (v/v), about 5% (v/v), about 10%(v/v), about 15% (v/v), about 20% (v/v), about 25% (v/v), about 30%(v/v), about 35% (v/v), about 40% (v/v), about 45% (v/v), or about 50%(v/v).

In some embodiments, the reaction conditions can comprise a surfactantfor stabilizing or enhancing the reaction. Surfactants can comprisenon-ionic, cationic, anionic and/or amphiphilic surfactants. Exemplarysurfactants, include by way of example and not limitations, nonylphenoxypolyethoxylethanol (NP40), Triton X-100,polyoxyethylene-stearylamine, cetyltrimethylammonium bromide, sodiumoleylamidosulfate, polyoxyethylene-sorbitanmonostearate,hexadecyldimethylamine, etc. Any surfactant that may stabilize orenhance the reaction may be employed. The concentration of thesurfactant to be employed in the reaction may be generally from 0.1 to50 mg/ml, particularly from 1 to 20 mg/ml.

In some embodiments, the reaction conditions can include an antifoamagent, which aid in reducing or preventing formation of foam in thereaction solution, such as when the reaction solutions are mixed orsparged. Anti-foam agents include non-polar oils (e.g., minerals,silicones, etc.), polar oils (e.g., fatty acids, alkyl amines, alkylamides, alkyl sulfates, etc.), and hydrophobic (e.g., treated silica,polypropylene, etc.), some of which also function as surfactants.Exemplary anti-foam agents include, Y-30® (Dow Corning), poly-glycolcopolymers, oxy/ethoxylated alcohols, and polydimethylsiloxanes. In someembodiments, the anti-foam can be present at about 0.001% (v/v) to about5% (v/v), about 0.01% (v/v) to about 5% (v/v), about 0.1% (v/v) to about5% (v/v), or about 0.1% (v/v) to about 2% (v/v). In some embodiments,the anti-foam agent can be present at about 0.001% (v/v), about 0.01%(v/v), about 0.1% (v/v), about 0.5% (v/v), about 1% (v/v), about 2%(v/v), about 3% (v/v), about 4% (v/v), or about 5% (v/v) or more asdesirable to promote the reaction.

The quantities of reactants used in the imine reductase reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of imine reductase substrate employed. Thosehaving ordinary skill in the art will readily understand how to varythese quantities to tailor them to the desired level of productivity andscale of production.

In some embodiments, the order of addition of reactants is not critical.The reactants may be added together at the same time to a solvent (e.g.,monophasic solvent, biphasic aqueous co-solvent system, and the like),or alternatively, some of the reactants may be added separately, andsome together at different time points. For example, the cofactor,co-substrate, imine reductase, and substrate may be added first to thesolvent.

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a pre-chilled lyophilizationchamber, followed by the application of a vacuum.

For improved mixing efficiency when an aqueous co-solvent system isused, the imine reductase, and cofactor may be added and mixed into theaqueous phase first. The organic phase may then be added and mixed in,followed by addition of the imine reductase substrate and co-substrate.Alternatively, the imine reductase substrate may be premixed in theorganic phase, prior to addition to the aqueous phase.

The imine reductase reaction is generally allowed to proceed untilfurther conversion of substrates to product does not changesignificantly with reaction time, e.g., less than 10% of substratesbeing converted, or less than 5% of substrates being converted). In someembodiments, the reaction is allowed to proceed until there is completeor near complete conversion of substrates to product. Transformation ofsubstrates to product can be monitored using known methods by detectingsubstrate and/or product, with or without derivatization. Suitableanalytical methods include gas chromatography, HPLC, and the like.

In some embodiments of the process, the suitable reaction conditionscomprise a loading of substrates of at least about 5 g/L, 10 g/L, 20g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L, or more, andwherein the method results in at least about 50%, 60%, 70%, 80%, 90%,95% or greater conversion of substrate compounds to product compound inabout 48 h or less, in about 36 h or less, or in about 24 h or less.

The engineered imine reductase polypeptides of the present inventionwhen used in the process under suitable reaction conditions result in adiastereomeric excess of the desired secondary or tertiary amine productin at least 90%, 95%, 96%, 97%, 98%, 99%, or greater. In someembodiments, no detectable amount of the undesired diastereomericsecondary or tertiary amine product is formed.

In further embodiments of the processes for converting substratecompounds to amine product compound using the engineered imine reductasepolypeptides, the suitable reaction conditions can comprise initialsubstrate loadings to the reaction solution which is then contacted bythe polypeptide. This reaction solution is the further supplemented withadditional substrate compounds as a continuous or batchwise additionover time at a rate of at least about 1 g/L/h, at least about 2 g/L/h,at least about 4 g/L/h, at least about 6 g/L/h, or higher. Thus,according to these suitable reaction conditions, polypeptide is added toa solution having initial ketone and amine substrate loadings of each atleast about 20 g/L, 30 g/L, or 40 g/L. This addition of polypeptide isthen followed by continuous addition of further ketone and aminesubstrates to the solution at a rate of about 2 g/L/h, 4 g/L/h, or 6g/L/h until a much higher final substrate loading of each at least about30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L, 150 g/L, 200 g/L ormore, is reached. Accordingly, in some embodiments of the process, thesuitable reaction conditions comprise addition of the polypeptide to asolution having initial substrate loadings of each at least about 20g/L, 30 g/L, or 40 g/L followed by addition of further ketone and aminesubstrates to the solution at a rate of about 2 g/L/h, 4 g/L/h, or 6g/L/h until a final substrate loading of at least about 30 g/L, 40 g/L,50 g/L, 60 g/L, 70 g/L, 100 g/L or more, is reached. These substratesupplementation reaction conditions allow for higher substrate loadingsto be achieved while maintaining high rates of conversion of substratesto amine product of at least about 50%, 60%, 70%, 80%, 90% or greaterconversion.

In some embodiments of the processes, the reaction using an engineeredimine reductase polypeptide can comprise the following suitable reactionconditions: (a) substrate loading at about 5 g/L to 30 g/L; (b) about0.1 g/L to 10 g/L of the engineered polypeptide; (c) about 19 g/L (0.13M) to 57 g/L (0.39 M) of α-ketoglutarate; (d) about 14 g/L (0.08 M) to63 g/L (0.36 M) ascorbic acid; (e) about 1.5 g/L (3.8 mM) to 4.5 g/L(11.5 mM) of FeSO₄; (f) a pH of about 6 to 9; (g) temperature of about20° to 50° C.; and (h) reaction time of 2-24 hrs.

In some embodiments of the processes, the reaction using an engineeredimine reductase polypeptide can comprise the following suitable reactionconditions: (a) substrate loading at about 10 g/L to 100 g/L; (b) about1 g/L to about 50 g/L of engineered polypeptide; (c) NADH or NADPHloading at about 0.1 g/L to about 5 g/L; (d) pH of about 6 to 10; (g)temperature of about 20° to 50° C.; and (h) reaction time of 6 to 120hrs.

In some embodiments, additional reaction components or additionaltechniques carried out to supplement the reaction conditions. These caninclude taking measures to stabilize or prevent inactivation of theenzyme, reduce product inhibition, shift reaction equilibrium to amineproduct formation.

In further embodiments, any of the above described process for theconversion of substrate compound to product compound can furthercomprise one or more steps selected from: extraction; isolation;purification; and crystallization of product compound. Methods,techniques, and protocols for extracting, isolating, purifying, and/orcrystallizing the amine product from biocatalytic reaction mixturesproduced by the above disclosed methods are known to the ordinaryartisan and/or accessed through routine experimentation. Additionally,illustrative methods are provided in the Examples below.

Various features and embodiments of the invention are illustrated in thefollowing representative examples, which are intended to beillustrative, and not limiting.

EXPERIMENTAL

The following examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention. In the experimental disclosure below,the following abbreviations apply: ppm (parts per million); M (molar);mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (moles); gmand g (gram); mg (milligrams); ug and μg (micrograms); L and 1 (liter);ml and mL (milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); U (units); MW (molecular weight); rpm (rotations per minute);psi and PSI (pounds per square inch); ° C. (degrees Centigrade); RT andrt (room temperature); CAM and cam (chloramphenicol); DMSO(dimethylsulfoxide); PMBS (polymyxin B sulfate); IPTG (isopropylβ-D-1-thiogalactopyranoside); LB (Luria broth); TB (terrific broth); SFP(shake flask powder); CDS (coding sequence); DNA (deoxyribonucleicacid); RNA (ribonucleic acid); E. coli W3110 (commonly used laboratoryE. coli strain, available from the Coli Genetic Stock Center [CGSC], NewHaven, Conn.); HTP (high throughput); HPLC (high pressure liquidchromatography); FIOPC (fold improvements over positive control);Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.; Difco (Difco Laboratories,BD Diagnostic Systems, Detroit, Mich.); Agilent (Agilent Technologies,Inc., Santa Clara, Calif.); Corning (Corning, Inc., Palo Alto, Calif.);Dow Corning (Dow Corning, Corp., Midland, Mich.); and Gene Oracle (GeneOracle, Inc., Mountain View, Calif.).

Example 1 Synthesis, Optimization, and Screening Engineered PolypeptidesDerived from CENDH Having Imine Reductase Activity

Gene synthesis and optimization: The polynucleotide sequence encodingthe reported wild-type opine dehydrogenase polypeptide CENDH fromArthrobacter sp. strain C1, as represented by SEQ ID NO:2, wascodon-optimized using the GeneIOS synthesis platform (GeneOracle) andsynthesized as the gene of SEQ ID NO:1. The synthetic gene of SEQ IDNO:1 was cloned into a pCK110900 vector system (See e.g., US Pat. Appln.Publn. No. 20060195947, which is hereby incorporated by referenceherein) and subsequently expressed in E. coli W3110fhuA. The E. colistrain W3110 expressed the opine dehydrogenase polypeptide CENDH underthe control of the lac promoter. Based on sequence comparisons withother CENDH (and other amino acid dehydrogenases) and computer modelingof the CENDH structure docked to the substrate, residue positionsassociated with the active site, peptide loops, solution/substrateinterface, and potential stability positions were identified.

Briefly, directed evolution of the CENDH gene was carried out byconstructing libraries of variant genes in which these positionsassociated with certain structural features were subjected tomutagenesis. These libraries were then plated, grown-up, and screenedusing HTP assays as described in Examples 2 and 3 to provide a firstround (“Round 1”) of 41 engineered CENDH variant polypeptides with iminereductase activity. The amino acid differences identified in these Round1 engineered CENDH variant polypeptides were recombined to build newRound 2 libraries which were then screened for activity with the ketonesubstrate of compound (1b) and the amine substrate of compound (2b) toproduce the secondary amine product compound (3d). This imine reductaseactivity screened for in Round 2 is not detectable in the naturallyoccurring opine dehydrogenase CENDH polypeptide from which the variantswere derived. Round 2 of directed evolution resulted in 7 engineeredpolypeptides having from 4 to 10 amino acid differences relative to SEQID NO:2 and the desired non-natural imine reductase activity. TheseRound 2 variants included SEQ ID NO:4, which has the 8 amino aciddifferences: X156T, X197I, X198E, X201L, X259H, X280L, X292V, X293H.Three further rounds of directed evolution starting with the engineeredpolypeptide of SEQ ID NO:4 were carried out and resulted in theengineered polypeptide of SEQ ID NO:6, which has at least 3-foldimproved imine reductase activity, relative to SEQ ID NO:4, inconverting the ketone substrate of compound (1j) and the amine substrateof compound (2b) to the secondary amine product compound (3o). Theengineered polypeptide of SEQ ID NO:6 has the following 22 additionalamino acid differences relative to the engineered polypeptide of SEQ IDNO:4 from which it was evolved: X29R, X94K, X111R, X137N, X157R, X184Q,X220H, X223T, X232A, X259V, X261I, X266T, X279V, X284M, X287T, X288S,X295S, X311V, X324L, X328E, X332V, and X353E. The engineered polypeptideof SEQ ID NO:6 was used as the starting “backbone” reference sequencefor further directed evolution of the various engineered polypeptides ofSEQ ID NOS:8-924 provided herein (See e.g., Tables 3A-3L).

Example 2 Production of Engineered Polypeptides Derived from CENDHHaving Imine Reductase Activity

The engineered imine reductase polypeptides of SEQ ID NOS:4-924 wereproduced in E. coli W3110 under the control of the lac promoter. Enzymepreparations for the HTP assays used in the directed evolution of theengineered polypeptides were made as follows.

High-Throughput (HTP) Growth, Expression, and Lysate Preparation.

Cells were picked and grown overnight in LB media containing 1% glucoseand 30 μg/mL CAM, 30° C., 200 rpm, 85% humidity. 20 μL of overnightgrowth were transferred to a deep well plate containing 380 μL TB growthmedia containing 30 μg/mL CAM, 1 mM IPTG, and incubated for ˜18 h at 30°C., 200 rpm, 85% humidity. Cell cultures were centrifuged at 4000 rpm,4° C. for 10 min., and the media discarded. Cell pellets thus obtainedwere stored at −80° C. and used to prepare lysate for HTP reactions asfollows. Lysis buffer containing 1 g/L lysozyme and 1 g/L PMBS wasprepared in 0.1 M phosphate buffer, pH 8.5 (or pH 10). Cell pellets in96 well plates were lysed in 250 μL lysis buffer, with low-speed shakingfor 1.5 h on a titre-plate shaker at room temperature. The plates thenwere centrifuged at 4000 rpm for 10 mins at 4° C. and the clearsupernatant was used as the clear lysate in the HTP assay reaction.

Production of Shake Flask Powders (SFP):

A shake-flask procedure can be used to generate engineered iminereductase polypeptide shake-flask powders (SFP) useful for secondaryscreening assays or which can be used to carry out the biocatalyticprocesses disclosed herein. Shake flask powder (SFP) preparation ofenzymes provides a more purified preparation (e.g., up to 30% of totalprotein) of the engineered enzyme as compared to the cell lysate used inHTP assays and, among other things, allows for the use of moreconcentrated enzyme solutions. A single colony of E. coli containing aplasmid encoding an engineered polypeptide of interest is inoculatedinto 50 mL Luria Bertani broth containing 30 μg/ml chloramphenicol and1% glucose. Cells are grown overnight (at least 16 hours) in anincubator at 30° C. with shaking at 250 rpm. The culture is diluted into250 mL Terrific Broth (12 g/L bacto-tryptone, 24 g/L yeast extract, 4mL/L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO₄) containing30 μg/ml CAM, in a 1 L flask to an optical density of 600 nm (0D600) of0.2 and allowed to grow at 30° C. Expression of the imine reductase geneis induced by addition of IPTG to a final concentration of 1 mM when theOD₆₀₀ of the culture is 0.6 to 0.8. Incubation is then continuedovernight (at least 16 hours). Cells are harvested by centrifugation(5000 rpm, 15 min, 4° C.) and the supernatant discarded. The cell pelletis resuspended with an equal volume of cold (4° C.) 50 mM potassiumphosphate buffer, pH 7.5, and harvested by centrifugation as above. Thewashed cells are resuspended in two volumes of the cold 50 mM potassiumphosphate buffer, pH 7.5 and passed through a French Press twice at12,000 psi while maintained at 4° C. Cell debris is removed bycentrifugation (10,000 rpm, 45 minutes, 4° C.). The clear lysatesupernatant is collected and stored at −20° C. Lyophilization of frozenclear lysate provides a dry shake-flask powder of crude engineeredpolypeptide. Alternatively, the cell pellet (before or after washing)can be stored at 4° C. or −80° C.

Example 3 Evolution and Screening of Engineered Polypeptides Derivedfrom SEQ ID NO:6 for Improved Stability and Imine Reductase Activity inPreparing Compounds (3n), (3o), (3p), (3q), (3r), and (3s)

The engineered polypeptide having imine reductase activity of SEQ IDNO:6 was used to generate further engineered polypeptides of Tables3A-3L which have further improved stability (e.g., activity at 44° C.,and 15% or 30% DMSO) and improved imine reductase activity (e.g., %conversion of ketone substrate compound (1j) to product). Theseengineered polypeptides, which have the amino acid sequences ofeven-numbered sequence identifiers SEQ ID NOS:8-924, were generated fromthe “backbone” amino acid sequence of SEQ ID NO:6 using directedevolution methods as described above together with the HTP assay andanalytical methods noted in Tables 3A-3L and described further below.

Directed evolution began with the polynucleotide of SEQ ID NO:5, whichencodes the engineered polypeptide of SEQ ID NO:6, as the starting“backbone” gene sequence. Libraries of engineered polypeptides weregenerated using various well-known techniques (e.g., saturationmutagenesis, recombination of previously identified beneficial aminoacid differences) and screened using HTP assay and analysis methods thatmeasured the ability of the engineered polypeptides to carry out one ormore of the catalytic reactions (o) through (s) shown in Table 2. Afterscreening, the engineered polypeptide(s) showing the most improvementover the starting backbone sequence (or “control” sequence) were used asbackbone sequences for the construction of further libraries, and thescreening process repeated to evolve the polypeptide for the desiredactivity. In particular, for catalytic reaction (p) the particularlyimproved backbone sequences included SEQ ID NOS:12, 92, and 350; forcatalytic reaction (q) the particularly improved backbone sequencesincluded SEQ ID NOS:12, 146, 350, and 440; for catalytic reaction (r)the particularly improved backbone sequences included SEQ ID NOS:12, 84,and 228; and for catalytic reaction (s) the particularly improvedbackbone sequences included SEQ ID NOS:12, 162, and 354.

Tables 3A-3L describe details of assays protocols and conditions used inevolving the engineered polypeptides of SEQ ID NOS:8-924 which areuseful for carrying the biocatalytic conversion reactions (a)-(s) ofTable 2, and in particular the biocatalytic reactions (o), (p), (q),(r), and (s), which produce the amine compound products (3o), (3p),(3q), (3r), and (3s). Further details of the analysis of these specificamine products generated in the biocatalytic assay mixtures is providedbelow.

HPLC Analysis for Amine Product Compound (3o) (Table 3A Assays):

The HTP assay mixtures prepared as noted in Table 3A were analyzed byHPLC using the instrument and parameters shown below.

Instrument Agilent HPLC 1200 series Column Onyx Monolithic C8, 100 × 4.5mm with Onyx monolithic C18 guard cartridge (Phenomenex) Mobile PhaseGradient (A: 0.1% formic acid in water; B: 0.1% formic acid in MeCN)Time (min) % B 0.0.-0.8 25 1.75 70 1.8-2.0 90 2.1 25 Flow Rate 2.0mL/min Run time 3.05 min Peak Retention Compound (3o): 1.17 min TimesCompound (1j): 2.17 min Column Temperature 40° C. Injection Volume 10 μLUV Detection 210 nm Detection Detector: MWD (Agilent 1200 series); Slit= 4 nm; peak width > 0.1 min; Reference = 360; BW = 8

LC-MS Analysis for Amine Product Compound (3p) (Table 3B and 3E Assays):

The HTP assay mixtures prepared as noted in Tables 3B and 3E wereanalyzed for formation of the product compound (3p) by LC-MS in MRM modeusing the MRM transition: 294/112. Additional relevant LC-MSinstrumental parameters and conditions were as shown below.

Instrument Agilent HPLC 1200 series, API 2000 Column Poroshell 120 ECC18 50 × 3.0 mm, 2.7 μm (Agilent Technologies) with ProShell 120 EC-C183.0 × 5 mm 2.7 micron guard column Mobile Phase Gradient (A: 0.1% formicacid in water; B: 0.1% formic acid in MeCN) Time (min) % B 0.0.-0.8 102.0-2.5 90 2.6-3.5 30 Flow Rate 0.8 mL/min Run time 3.5 min PeakRetention Times Compound (3p): 2.59 min Column Temperature RoomTemperature Injection Volume 10 μL MS Detection API 2000; MRM294/112(3p); MS Conditions MODE: MRM; CUR: 30; IS: 4500; CAD 6: TEM: 550° C.;GS1: 60; GS2: 60; DP: 31; FP: 350; EP: 10; CE: 30; CXP: 4; DT: 100 ms

HPLC Analysis for Amine Product Compound (3p) (Table 3I Assays):

The HTP assay mixtures prepared as noted in Table 3I were analyzed forformation of the product compound (3p) by HPLC using the instrumentalparameters and conditions shown below.

Instrument Agilent HPLC 1200 series Column Onyx Monolithic C8, 100 × 4.5mm with Onyx monolithic C18 guard cartridge (Phenomenex) Mobile PhaseGradient (A: 0.1% formic acid in water; B: 0.1% formic acid in MeCN)Time (min) % B 0.0.-0.8 25 1.75 70 1.8-2.0 90 2.1 25 Flow Rate 2.0mL/min Run time 3.05 min Peak Retention Times Compound (3p): 1.18 minCompound (1j): 2.12 min Column Temperature 40° C. Injection Volume 10 μLUV Detection 210 nm Detection Detector: MWD (Agilent 1200 series); Slit= 4 nm; peak width > 0.1 min; Reference = 360; BW = 8

HPLC Analysis for Amine Product Compound (3q) (Table 3B, 3C, 3G and 3KAssays):

The HTP assay mixtures prepared as noted in Tables 3B, 3C, 3G and 3Kwere analyzed for formation of the product compound (3q) by HPLC usingthe instrumental parameters and conditions shown below.

Instrument Agilent HPLC 1200 series Column Onyx Monolithic C8, 100 × 4.5mm with Onyx monolithic C18 guard cartridge (Phenomenex) Mobile PhaseGradient (A: 0.1% formic acid in water; B: 0.1% formic acid in MeCN)Time (min) % B 0.0.-0.8 25 1.75 70 1.8-2.0 90 2.1 25 Flow Rate 2.0mL/min Run time 3.05 min Peak Retention Times Compound (3q): 1.17 minCompound (1j): 2.17 min Column Temperature 40° C. Injection Volume 10 μLUV Detection 210 nm Detection Detector: MWD (Agilent 1200 series); Slit= 4 nm; peak width > 0.1 min; Reference = 360; BW = 8

LC-MS Analysis for Amine Product Compound (3r) (Table 3B, and 3DAssays):

The HTP assay mixtures prepared as noted in Tables 3B and 3D wereanalyzed using LC-MS for formation of the product compound (3r),N-propyl-5-methoxy-1,2,3,4-tetrahydronaphthalen-2-amine, using the MRMtransition: 294/112. Additional relevant LC-MS instrumental parametersand conditions were as shown below

Instrument Agilent HPLC 1200 series, API 2000 Column Poroshell 120 ECC18 50 × 3.0 mm, 2.7 μm (Agilent Technologies) with ProShell 120 EC-C183.0 × 5 mm 2.7 micron guard column Mobile Phase Gradient (A: 0.1% formicacid in water; B: 0.1% formic acid in MeCN) Time (min) % B 0.0.-0.8 251.11 44 1.20-1.70 90 1.80-2.50 30 Flow Rate 0.8 mL/min Run time 2.5 minPeak Retention Times Compound (3r): 0.55 min Column Temperature RoomTemperature Injection Volume 10 μL MS Detection API 2000; MRM220/161(for N-propy1-5-methoxy-1,2,3,4- tetrahydronaphthalen-2-amine); MSConditions MODE: MRM; CUR: 30; IS: 4500; CAD 6: TEM: 550° C.; GS1: 60;GS2: 60; DP: 31; FP: 350; EP: 10; CE: 25; CXP: 6; DT: 100 ms

HPLC Analysis for Amine Product Compound (3r) (Table 3H Assay):

The HTP assay mixtures prepared as noted in Table 3H were analyzed forformation of the product compound (3r) by HPLC using the instrumentalparameters and conditions shown below.

Instrument Agilent HPLC 1200 series Column Onyx Monolithic C8, 100 × 4.5mm with Onyx monolithic C18 guard cartridge (Phenomenex) Mobile PhaseGradient (A: 0.1% formic acid in water; B: 0.1% formic acid in MeCN)Time (min) % B 0.0.-0.8 25 1.75 75 1.8-2.0 90 2.1 25 Flow Rate 2.0mL/min Run time 3.05 min Peak Retention Times Compound (3r): 1.11 minCompound (1i): 2.06 min Column Temperature 40° C. Injection Volume 10 μLUV Detection 210 nm Detection Detector: MWD (Agilent 1200 series); Slit= 4 nm; peak width > 0.1 min; Reference = 360; BW = 8

LC-MS Analysis for Amine Product Compound (3s) (Table 3B, 3F, 3J and 3LAssays):

The HTP assay mixtures prepared as noted in Tables 3B, 3F, 3J and 3Lwere analyzed using LC-MS for formation of the product compound (3s)using the MRM transition: 206/174. Additional relevant LC-MSinstrumental parameters and conditions were as shown below.

Instrument Agilent HPLC 1200 series, API 2000 Column Poroshell 120 ECC18 50 × 3.0 mm, 2.7 μm (Agilent Technologies) with ProShell 120 EC-C183.0 × 5mm 2.7 micron guard column Mobile Phase Gradient (A: 0.1% formicacid in water; B: 0.1% formic acid in MeCN) Time (min) % B 0.0.-0.8 251.11 44 1.20-1.70 90 1.80-2.50 30 Flow Rate 0.8 mL/min Run time 2.5 minPeak Retention Times Compound (3s): 0.77 min Column Temperature RoomTemperature Injection Volume 10 μL MS Detection API 2000; MRM206/174(3s); MS Conditions MODE: MRM; CUR: 30; IS: 4500; CAD 12: TEM: 550° C.;GS1: 60; GS2: 60; DP: 31; FP: 350; EP: 10; CE: 20; CXP: 4; DT: 100 ms

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. An engineered polypeptide comprising an aminoacid sequence with at least 80% sequence identity to a referencesequence of SEQ ID NO:6 and at least one of the following features: (i)a residue difference as compared to the reference sequence of SEQ IDNO:6 at a position selected from X12, X18, X26, X27, X57, X65, X87, X93,X96, X126, X138, X140, X142, X159, X170, X175, X177, X195, X200, X221,X234, X241, X242, X253, X254, X257, X262, X263, X267, X272, X276, X277,X278, X281, X282, X291, and X352, optionally wherein the residuedifference at the position is selected from X12M, X18G, X26M/V, X27S,X57D/L/V, X65I/V, X87A, X93G/Y, X96C, X126S, X138L, X140M, X142A,X159C/L/Q/V, X170F/K/R/S, X175R, X177R, X195S, X200S, X221F, 234C/L,X241K, X242C/L, X253K/N, X254R, X257Q, X262F/G/P/V,X263C/D/E/H/I/K/L/M/N/P/Q/V, X267E/G/H/I/N/S, X272D, X276L, X277H/L,X278E/H/K/N/R/S/W, X281A, X282A/R, X291E, and X352Q; (ii) a residuedifference as compared to the reference sequence of SEQ ID NO:6 selectedfrom X20V, X29K, X37P, X74W, X82C/T, X94N, X108S, X111A/H, X141M/N,X143F/L/Y, X153E/F, X154C/D/G/K/L/N/S/T/V, X156H/L/N/M/R, X157F/Q/T/Y,X158I/L/R/S/T/V, X163V, X197V, X201I, X220C/K/Q, X223S, X256A/E/I/L/S/T,X259C/R, X260A/D/N/Q/V/Y, X261E/F/H/L/P/Q/Y, X264V, X270L, X273C,X274L/S, X279T, X284C/F/H/P/Q/S, X292E/P, and X295F; and/or (iii) two ormore residue differences as compared to the reference sequence of SEQ IDNO:6 selected from X153Y, X283V, X82P, X141W, X154F, X259I/L/M, X274L/M,and X296N/V; wherein the polypeptide has imine reductase activity. 2.The engineered polypeptide of claim 1, wherein the amino acid sequencecomprises at least one residue difference as compared to the referencesequence of SEQ ID NO:6 selected from X12M, X37P, X82T, X111A, X154S,X156N/M, X223S, X256E, X260D, X261H, X262P, X263C/E/Q, X267G, X277L,X281A, X284P/S, and X292E.
 3. The engineered polypeptide of claim 1,wherein the amino acid sequence comprises at least one residuedifference as compared to the reference sequence of SEQ ID NO:6 selectedfrom X256E, X93G/Y, X94N, X96C, X111A/H, X142A, X159L, X163V, X259R,X273C, and X284P/S.
 4. The engineered polypeptide of claim 1, whereinthe amino acid sequence comprises at least two residue differences ascompared to the reference sequence of SEQ ID NO:6 selected fromX82C/P/T, X141W, X143W, X153Y, X154F/Q/Y, X256V, X259I/L/M/T, X260G,X261R, X265L, X273W, X274M, X277A/I, X279L, X283V, X284L, X296N, X326V.5. The engineered polypeptide of claim 1, wherein the amino acidsequence comprises at least a combination of residue differences ascompared to the reference sequence of SEQ ID NO:6 selected from: (a)X153Y, and X283V; (b) X141W, X153Y, and X283V; (c) X141W, X153Y,X274L/M, and X283V; (d) X141W, X153Y, X154F, X274L/M, and X283V; (e)X141W, X153Y, X154F, and X283V; (f) X141W, X153Y, X283V, and X296N/V;(g) X141W, X153Y, X274L/M, X283V, and X296N/V: (h) X111A, X153Y, X256E,X274M, and X283V; (i) X111A, X141W, X153Y, X273C, X274M, X283V, andX284S; (j) X111A, X141W, X153Y, X273C, and X283V; (k) X111A, X141W,X153Y, X154F, X256E, X274M, X283V, X284S, and X296N; (l) X111A, X141W,X153Y, X256E, X273W, X274L, X283V, X284S, and X296N; (m) X111H, X141W,X153Y, X273W, X274M, X284S, and X296N; (n) X111H, X141W, X153Y, X154F,X273W, X274L, X283V, X284S, and X296N; (o) X82P, X141W, X153Y, X256E,X274M, and X283V; (p) X82P, X111A, X141W, X153Y, X256E, X274M, X283V,M284S, and E296V; (q) X94N, X143W, X159L, X163V, X259M, and X279L; (r)X141W, X153Y, X154F, and X256E; and (s) X153Y, X256E, and X274M.
 6. Theengineered polypeptide of claim 5, wherein the amino acid sequencefurther comprises at least one residue difference as compared to thereference sequence of SEQ ID NO:6 selected from X12M, X18G, X20V,X26M/V, X27S, X29K, X37P, X57D/L/V, X65I/V, X74W, X82C/T, X87A, X93G/Y,X94N, X96C, X108S, X111A/H, X126S, X138L, X140M, X141M/N, X142A,X143F/L/Y, X153E/F, X154C/D/G/K/L/N/S/T/V, X156H/L/N/M/R, X157F/Q/T/Y,X158I/L/R/S/T/V, X159C/L/Q/V, X163V, X170F/K/R/S, X175R, X177R, X195S,X197V, X200S, X201I, X220C/K/Q, X221F, X223S, X234V/C/L, X241K, X242C/L,X253K/N, X254R, X256A/E/I/L/S/T, X257Q, X259C/R, X260A/D/N/Q/V/Y,X261E/F/H/L/P/Q/Y, X262P, X262F/G/V, X263C/D/E/H/I/K/L/M/N/P/Q/V, X264V,X267E/G/H/I/N/S, X270L, X272D, X273C, X274L/S, X276L, X277H/L,X278E/H/K/N/R/S/W, X279T, X281A, X282A/R, X284C/F/H/P/Q/S, X291E,X292E/P, X295F, and X352Q.
 7. The engineered polypeptide of claim 5,wherein the amino acid sequence further comprises at least one residuedifference as compared to the reference sequence of SEQ ID NO:6 selectedfrom X12M, X37P, X82T, X111A, X141W, X153Y, X154 F/S, X156N/M, X223S,X256E, X259I, X260D, X261H, X262P, X263C/E/Q, X267G, X274M, X277L,X281A, X283V, X284P/S, X292E, and X296N.
 8. The engineered polypeptideof claim 1, wherein the amino acid sequence comprises the residuedifferences X111A, X141W, X153Y, X154F, X256E, X274M, X283V, X284S, andX296N and at least residue difference or a combination of residuedifferences as compared to the reference sequence of SEQ ID NO:6selected from: (a) X156N; (b) X37P, X82T, and X156N; (c) X37P, X82T,X156N, and X259I; (d) X259L/M; (e) X82T, X156N, X223S, X259L, X267G, andX281A; (f) X263C; (g) X12M, X261H, X263C, X277L, and X292E; (h) X154S;and (i) X154S, X156M, X260D, X261H, X262P, X263E, and X284P.
 9. Theengineered polypeptide of claim 1, wherein the amino acid sequence doesnot include a residue difference as compared to the reference sequenceof SEQ ID NO:6 at a residue position selected from X29, X137, X157,X184, X197, X198, X201, X220, X232, X261, X266, X279, X280, X287, X288,X293, X295, X311, X324, X328, X332, and X353.
 10. The engineeredpolypeptide of claim 1, wherein the amino acid sequence furthercomprises a residue difference as compared to the reference sequence ofSEQ ID NO:6 selected from: X4H/L/R, X5T, X14P, X20T, X29R/T, X37H,X67A/D, X71C/V, X74R, X82P, X94K/R/T, X97P, X100W, X111M/Q/R/S, X124L/N,X136G, X137N, X141W, X143W, X149L, X153E/V/Y, X154F/M/Q/Y,X156G/I/Q/S/T/V, X157D/H/L/M/N/R, X158K, X160N, X163T, X177C/H, X178E,X183C, X184K/Q/R, X185V, X186K/R, X197I/P, X198A/E/H/P/S, X201L,X220D/H, X223T, X226L, X232G/A/R, X243G, X246W, X256V, X258D,X259E/H/I/L/M/S/T/V/W, X260G, X261A/G/I/K/R/S/T, X265G/L/Y, X266T,X270G, X273W, X274M, X277A/I, X279F/L/V/Y, X280L, X283M/V, X284K/L/M/Y,X287S/T, X288G/S, X292C/G/I/P/S/T/V/Y, X293H/I/K/L/N/Q/T/V, X294A/I/V,X295R/S, X296L/N/V/W, X297A, X308F, X311C/T/V, X323C/I/M/T/V, X324L/T,X326V, X328A/G/E, X332V, X353E, and X356R.
 11. The engineeredpolypeptide of claim 1, wherein the amino acid sequence does not includea residue difference as compared to the reference sequence of SEQ IDNO:6 at the following residue positions X29, X137, X157, X184, X197,X198, X201, X220, X232, X261, X266, X279, X280, X287, X288, X293, X295,X311, X324, X328, X332, and X353.
 12. The engineered polypeptide ofclaim 1, wherein the imine reductase activity comprises converting atleast one of the following ketone and amine substrate compound pairs tothe listed amine product compound under suitable reaction conditions:(a) ketone substrate compound (1j)


13. The engineered polypeptide of claim 12, wherein the imine reductaseactivity in converting the ketone and amine substrate compound pair tothe listed amine product compound under suitable reaction conditions isincreased at least 2-fold as compared to the corresponding activity ofthe reference polypeptide of SEQ ID NO:6.
 14. The engineered polypeptideof claim 1, in which the amino acid sequence comprises a sequenceselected from the group consisting of the even-numbered sequenceidentifiers of SEQ ID NOS:8-924.
 15. A polynucleotide encoding theengineered polypeptide of claim
 1. 16. The polynucleotide of claim 15,in which the polynucleotide comprises a nucleic acid sequence selectedfrom the group consisting of the odd-numbered sequence identifiers ofSEQ ID NOS:7-923.
 17. An expression vector comprising the polynucleotideof claim
 15. 18. A host cell comprising the polynucleotide of claim 15.19. A host cell comprising the expression vector of claim
 17. 20. Amethod of preparing the engineered polypeptide having imine reductaseactivity, comprising culturing the host cell of claim 18, underconditions suitable for expression of the polypeptide, optionallyfurther comprising isolating the engineered polypeptide.